Daric interleukin receptors

ABSTRACT

The present disclosure provides improved compositions for adoptive T cell therapies for treating, preventing, or ameliorating at least one symptom of a cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency, or condition associated therewith.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/598,923, filed Dec. 14, 2017, which isincorporated by reference herein in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is BLBD_092_01WO_ST25.txt. The text file is 28 KB,was created on Dec. 14, 2018, and is being submitted electronically viaEFS-Web, concurrent with the filing of the specification.

BACKGROUND Technical Field

The present disclosure relates to improved adoptive cell therapies. Moreparticularly, the disclosure relates to improved chemically regulatedsignaling molecules, cells, and methods of using the same for modulatingspatial and temporal control of cellular signal initiation anddownstream responses during adoptive immunotherapy.

Description of the Related Art

The global burden of cancer doubled between 1975 and 2000. Cancer is thesecond leading cause of morbidity and mortality worldwide, withapproximately 14.1 million new cases and 8.2 million cancer relateddeaths in 2012. The most common cancers are breast cancer, lung andbronchus cancer, prostate cancer, colon and rectum cancer, bladdercancer, melanoma of the skin, non-Hodgkin lymphoma, thyroid cancer,kidney and renal pelvis cancer, endometrial cancer, leukemia, andpancreatic cancer. The number of new cancer cases is projected to riseto 22 million within the next two decades.

Adoptive cellular therapy is emerging as a powerful paradigm fordelivering complex biological signals to treat cancer. In contrast tosmall molecule and biologic drug compositions, adoptive cell therapieshave the potential to execute unique therapeutic tasks owing to theirmyriad sensory and response programs and increasingly defined mechanismsof genetic control. To achieve such therapeutic value, cells need to beoutfitted with machinery for sensing and integrating chemical and/orbiological information associated with local physiological environments.

BRIEF SUMMARY

The present disclosure generally relates, in part, to IL DARICcompositions, polynucleotides, polypeptides, and methods of making andusing the same.

In various embodiments, the present disclosure contemplates, in part, afusion polypeptide comprising: a first polypeptide comprising a firstmultimerization domain, a first transmembrane domain, and a first immunereceptor intracellular signaling domain; a polypeptide cleavage signal;and a second polypeptide comprising a second multimerization domain, asecond transmembrane domain; and a second immune receptor intracellularsignaling domain.

In certain embodiments, the first multimerization domain and the secondmultimerization domain are the same.

In some embodiments, the first multimerization domain and the secondmultimerization domain are different.

In some embodiments, the first multimerization domain and the secondmultimerization domain associate with a bridging factor selected fromthe group consisting of: rapamycin or a rapalog thereof, coumermycin ora derivative thereof, gibberellin or a derivative thereof, abscisic acid(ABA) or a derivative thereof, methotrexate or a derivative thereof,cyclosporin A or a derivative thereof, FK506/cyclosporin A (FKCsA) or aderivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506binding protein (FKBP) (SLF) or a derivative thereof.

In particular embodiments, the first multimerization domain and thesecond multimerization domain are a pair selected from the groupconsisting of: FKBP and FKBP12-rapamycin binding (FRB) or variantsthereof; FKBP and calcineurin or variants thereof; FKBP and cyclophilinor variants thereof; FKBP and bacterial dihydrofolate reductase (DHFR)or variants thereof; calcineurin and cyclophilin or variants thereof;PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1) or variantsthereof; and GIB1 and GAI or variants thereof.

In various embodiments, the first multimerization domain comprises aFKBP polypeptide or variant thereof, and the second multimerizationdomain comprises a FRB polypeptide or variant thereof.

In certain embodiments, the first multimerization domain comprises a FRBpolypeptide or variant thereof, and the second multimerization domaincomprises a FKBP polypeptide or variant thereof.

In particular embodiments, the first and second multimerization domainsare selected from FRB T2098L and FKBP12; and the bridging factor isAP21967.

In particular embodiments, the bridging factor is selected from thegroup consisting of: AP21967, sirolimus, everolimus, novolimus,pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, andzotarolimus.

In some embodiments, the first transmembrane domain and the secondtransmembrane domain are independently selected from the groupconsisting of: a CD4 transmembrane domain, a CD8a transmembrane domain,an amnionless (AMN) transmembrane domain, a CD28 transmembrane domain, aCD154 transmembrane domain, and a CD71 transmembrane domain.

In certain embodiments, the first transmembrane domain and the secondtransmembrane domain are independently selected from the groupconsisting of: a CD4 transmembrane domain and a CD8a transmembranedomain.

In some embodiments, the first transmembrane domain and the secondtransmembrane domain are the same.

In various embodiments, the first transmembrane domain and the secondtransmembrane domain are different.

In certain embodiments, the first immune receptor intracellularsignaling domain and the second immune receptor intracellular signalingdomain are isolated from a cytokine receptor, an interleukin receptor, apattern recognition receptor, or a toll-like receptor.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IL-12Rβ2 intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-12Rβ1 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IL-12Rβ1 intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-12Rβ2 intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an IL-7Rα intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an IL-2Rγintracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-7Rα intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an IL-2Rβ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rγ intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rβ intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an IL-21R intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an IL-2Rγintracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-21R intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IL-18R1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-18RAP intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an IL-18RAP intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-18R1 intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an IL-1R1 intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an IL-1RAPintracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an IL-1RAP intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1R1 intracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an IL-1RL2 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1RAP intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an IL-1RAP intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1RL2 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IFNAR1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIFNAR2 intracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an IFNAR2 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIFNAR1 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an TLR1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR1 intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an TLR2 intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an TLR2intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an TLR3 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR3 intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an TLR4 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR4 intracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an TLR5 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR5 intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an TLR6 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR6 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an TLR7 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR7 intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an TLR8 intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an TLR8intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an TLR9 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR9 intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an TLR10 intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an TLR10intracellular signaling domain.

In various embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide.

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving 2A polypeptide.

In certain embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide selected from the group consisting of: afoot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis Avirus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, aporcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide,and an encephalomyocarditis virus 2A peptide.

In certain embodiments, the first multimerization domain localizesextracellularly when the first polypeptide is expressed and the secondmultimerization domain localizes extracellularly when the secondpolypeptide is expressed.

In particular embodiments, a composition comprising a fusion polypeptidecontemplated herein is provided.

In various embodiments, the present disclosure contemplates, in part, acomposition comprising a first polypeptide comprising a firstmultimerization domain, a first transmembrane domain, and a first immunereceptor intracellular signaling domain; and a second polypeptidecomprising a second multimerization domain, a second transmembranedomain, and a second immune receptor intracellular signaling domain.

In some embodiments, the first multimerization domain and the secondmultimerization domain are the same.

In various embodiments, the first multimerization domain and the secondmultimerization domain are different.

In particular embodiments, the first multimerization domain and thesecond multimerization domain associate with a bridging factor selectedfrom the group consisting of: rapamycin or a rapalog thereof,coumermycin or a derivative thereof, gibberellin or a derivativethereof, abscisic acid (ABA) or a derivative thereof, methotrexate or aderivative thereof, cyclosporin A or a derivative thereof,FK506/cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim(Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or aderivative thereof.

In various embodiments, the first multimerization domain and the secondmultimerization domain are a pair selected from the group consisting of:FKBP and FKBP12-rapamycin binding (FRB) or variants thereof; FKBP andcalcineurin or variants thereof; FKBP and cyclophilin or variantsthereof; FKBP and bacterial dihydrofolate reductase (DHFR) or variantsthereof; calcineurin and cyclophilin or variants thereof; PYR1-like 1(PYL1) and abscisic acid insensitive 1 (ABI1) or variants thereof; andGIB1 and GAI or variants thereof.

In particular embodiments, the first multimerization domain comprises aFKBP polypeptide or variant thereof, and the second multimerizationdomain comprises a FRB polypeptide or variant thereof.

In some embodiments, the first multimerization domain comprises a FRBpolypeptide or variant thereof, and the second multimerization domaincomprises a FKBP polypeptide or variant thereof.

In certain embodiments, the first and second multimerization domains areselected from FRB T2098L and FKBP12; and the bridging factor is AP21967.

In various embodiments, the bridging factor is selected from the groupconsisting of: AP21967, sirolimus, everolimus, novolimus, pimecrolimus,ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

In various embodiments, the first transmembrane domain and the secondtransmembrane domain are independently selected from the groupconsisting of: a CD4 transmembrane domain, a CD8a transmembrane domain,an amnionless (AMN) transmembrane domain, a CD28 transmembrane domain, aCD154 transmembrane domain, and a CD71 transmembrane domain.

In some embodiments, the first transmembrane domain and the secondtransmembrane domain are independently selected from the groupconsisting of: a CD4 transmembrane domain and a CD8a transmembranedomain.

In certain embodiments, the first transmembrane domain and the secondtransmembrane domain are the same.

In various embodiments, the first transmembrane domain and the secondtransmembrane domain are different.

In particular embodiments, first immune receptor intracellular signalingdomain and the second immune receptor intracellular signaling domain areisolated from a cytokine receptor, an interleukin receptor, a patternrecognition receptor, or a toll-like receptor.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an IL-12Rβ2 intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-12Rβ1 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IL-12Rβ1 intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-12Rβ2 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IL-7Rα intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rγ intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an IL-2Rγ intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an IL-7Rαintracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an IL-2Rβ intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an IL-2Rγintracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rβ intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an IL-21R intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rγ intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-21R intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an IL-18R1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-18RAP intracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an IL-18RAP intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-18R1 intracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an IL-1R1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1RAP intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IL-1RAP intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1R1 intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an IL-1RL2 intracellular signaling domain and thesecond immune receptor intracellular signaling domain comprises anIL-1RAP intracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an IL-1RAP intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1RL2 intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an IFNAR1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIFNAR2 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an IFNAR2 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIFNAR1 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an TLR1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR1 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an TLR2 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR2 intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an TLR3 intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an TLR3intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an TLR4 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR4 intracellular signaling domain.

In certain embodiments, the first immune receptor intracellularsignaling domain comprises an TLR5 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR5 intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an TLR6 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR6 intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an TLR7 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR7 intracellular signaling domain.

In some embodiments, the first immune receptor intracellular signalingdomain comprises an TLR8 intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an TLR8intracellular signaling domain.

In particular embodiments, the first immune receptor intracellularsignaling domain comprises an TLR9 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR9 intracellular signaling domain.

In various embodiments, the first immune receptor intracellularsignaling domain comprises an TLR10 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR10 intracellular signaling domain.

In particular embodiments, the composition further comprises a cell.

In certain embodiments, a polypeptide complex comprising a first andsecond polypeptide contemplated herein is provided.

In particular embodiments, a polynucleotide encoding a fusionpolypeptide or a first and second polypeptide contemplated herein isprovided.

In various embodiments, a cDNA encoding a fusion polypeptide or a firstand second polypeptide contemplated herein is provided.

In some embodiments, an RNA encoding a fusion polypeptide or a first andsecond polypeptide contemplated herein is provided.

In various embodiments, vector comprising a polynucleotide contemplatedherein is provided.

In certain embodiments, a cell comprising a fusion polypeptide, a firstand second polypeptide, a polynucleotide, or a vector contemplatedherein is provided.

In particular embodiments, the cell is a hematopoietic cell.

In various embodiments, the cell is a T cell.

In some embodiments, the cell is a CD3+, CD4+, and/or CD8+ cell.

In various embodiments, the cell is an immune effector cell.

In particular embodiments, the cell is a cytotoxic T lymphocytes (CTLs),a tumor infiltrating lymphocytes (TILs), or a helper T cells.

In some embodiments, the cell is a natural killer (NK) cell or naturalkiller T (NKT) cell.

In particular embodiments, the source of the cell is peripheral bloodmononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymusissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, or tumors.

In various embodiments, the cell further comprises an engineered antigenreceptor.

In certain embodiments, the engineered antigen receptor is selected fromthe group consisting of: an engineered T cell receptor (TCR), a chimericantigen receptor (CAR), a DARIC receptor or components thereof, and achimeric cytokine receptor.

In various embodiments, a composition comprising a fusion polypeptide, afirst and second polypeptide, a polynucleotide, a vector, or a cellcontemplated herein is provided.

In various embodiments, a pharmaceutical composition comprising apharmaceutically acceptable carrier and a composition comprising afusion polypeptide, a first and second polypeptide, a polynucleotide, avector, or a cell contemplated herein is provided.

In various embodiments, a method of treating a subject in need thereofcomprising administering the subject an effective amount of acomposition contemplated herein is provided.

In various embodiments, a method of treating, preventing, orameliorating at least one symptom of a cancer, infectious disease,autoimmune disease, inflammatory disease, and immunodeficiency, orcondition associated therewith, comprising administering to the subjectan effective amount of a composition contemplated herein is provided.

In various embodiments, a method of treating a solid cancer comprisingadministering to the subject an effective amount of a compositioncontemplated herein is provided.

In particular embodiments, the solid cancer comprises liver cancer,pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostatecancer, testicular cancer, bladder cancer, brain cancer, sarcoma, headand neck cancer, bone cancer, thyroid cancer, kidney cancer, or skincancer.

In certain embodiments, the solid cancer is a pancreatic cancer, a lungcancer, or a breast cancer.

In various embodiments, the present disclosure contemplates, in part, amethod of treating a hematological malignancy comprising administeringto the subject an effective amount of a composition contemplated herein.

In particular embodiments, the hematological malignancy is a leukemia,lymphoma, or multiple myeloma.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A shows a cartoon of IL-18R DARIC and IL-18R DARIC-GFP constructsand polypeptides.

FIG. 1B shows GFP expression in donor PBMCs transduced with LVV encodingIL-18R-DARIC-GFP.

FIG. 1C shows growth curves for untransduced donor PBMCs and PBMCstransduced with LVV encoding IL-18R-DARIC-GFP.

FIG. 1D shows CD62L and CD45Ra expression in untransduced donor PBMCsand PBMCs transduced with LVV encoding IL-18R-DARIC-GFP.

FIG. 2 shows IFNγ production in untransduced donor PBMCs and PBMCstransduced with LVV encoding IL-18R-DARIC-GFP and pre-treated with 50ng/mL human recombinant IL-12 for 24 hours, and then subsequentlycultured in medium only, medium with 100 ng/mL human recombinant IL-18or medium with 1 nM rapamycin.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

SEQ ID NO: 1 sets forth the amino acid sequence for an IL-18R DARICsignaling component.

SEQ ID NO: 2 sets forth the amino acid sequence for an IL-18R DARICsignaling component.

SEQ ID NO: 3 sets forth the amino acid sequence for an IL-18R DARICbinding component.

SEQ ID NO: 4 sets forth the amino acid sequence for an IL-18R DARICbinding component.

SEQ ID NO: 5 sets forth the amino acid sequence for an IL-18R DARICpolyprotein comprising an IL-18R DARIC binding component and signalingcomponent separated by a viral P2A domain.

SEQ ID NO: 6 sets forth the amino acid sequence for an IL-18R DARIC-GFPpolyprotein comprising an IL-18R DARIC binding component, signalingcomponent, and

GFP, each separated by a viral P2A domain.

SEQ ID NOs: 7-17 set forth the amino acid sequences of various linkers.

SEQ ID NOs: 18-42 set forth the amino acid sequences of proteasecleavage sites and self-cleaving polypeptide cleavage sites.

DETAILED DESCRIPTION A. Overview

The present disclosure generally relates to chemically regulatablepolypeptides that transduce immunostimulatory signals to cellsexpressing the polypeptides. Without wishing to be bound by anyparticular theory, the polypeptides contemplated herein are fusionpolypeptides comprising a chemically inducible multimerization domainlinked to one or more immunostimulatory endodomains that stimulateimmune effector cell activity and function. Coexpression ofimmunostimulatory fusion polypeptides in immune effector cells in thepresence of a factor that induces multimerization and activation of thefusion polypeptides renders the cells resistant to the immunosuppressiveimpacts of the TME by restoring or increasing proinflammatory cytokinesecretion. In particular preferred embodiments, a fusion polypeptide isreferred to as a DARIC immune receptor.

In various embodiments, the present disclosure contemplates, in part,polypeptides that convert a chemical signal to an immunostimulatorysignal mediated through or by multimerization of intracellular domainsof immune receptors.

In various embodiments, the present disclosure contemplates, in part,polypeptides that convert a chemical signal to an immunostimulatorysignal mediated through or by multimerization of intracellular domainsof cytokine receptors.

In various embodiments, the present disclosure contemplates, in part,polypeptides that convert a chemical signal to an immunostimulatorysignal mediated through or by multimerization of intracellular domainsof interleukin receptors.

In various embodiments, the present disclosure contemplates, in part,polypeptides that convert a chemical signal to an immunostimulatorysignal mediated through or by multimerization of intracellular domainsof pattern recognition receptors.

In various embodiments, the present disclosure contemplates, in part,polypeptides that convert a chemical signal to an immunostimulatorysignal mediated through or by multimerization of intracellular domainsof toll-like receptors.

In particular embodiments, the present disclosure contemplates, in part,a polypeptide comprising an inducible multimerization domain, atransmembrane domain and one or more intracellular domains of one ormore immune receptors; and a polypeptide comprising another induciblemultimerization domain, a transmembrane domain and one or moreintracellular domains of one or more immune receptors. In oneembodiment, the polypeptides are linked to each other by a polypeptidecleavage signal, e.g., a 2A polypeptide cleavage signal.

In particular embodiments, the present disclosure contemplates, in part,an immune effector cell, e.g., CAR T cell or engineered TCR T cell, thatexpresses a polypeptide comprising an inducible multimerization domain,a transmembrane domain and one or more intracellular domains of one ormore immune receptors; and a polypeptide comprising another induciblemultimerization domain, a transmembrane domain and one or moreintracellular domains of one or more immune receptors.

In particular embodiments, the transmembrane domains are isolated from areceptor expressed on an immune effector cell; and intracellularsignaling domains are isolated from an IL-12 receptor, an IL-7 receptor,an IL-15 receptor, an IL-21 receptor, an IL-2 receptor, an IL-1receptor, an IL-18 receptor, an IL-36 receptor, a type I IFN receptor, aTLR1 receptor, a TLR2 receptor, a TLR3 receptor, a TLR4 receptor, a TLR5receptor, a TLR6 receptor, a TLR7 receptor, a TLR8 receptor, a TLR9receptor, or a TLR10 receptor.

In particular embodiments, the intracellular signaling domains areisolated from IL-12Rβ2, IL-7Rα, IL-2Rγ, IL-2R13, IL-21R, IL-18R1,IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2, IL-1RL2, TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10.

Techniques for recombinant (i.e., engineered) DNA, peptide andoligonucleotide synthesis, immunoassays, tissue culture, transformation(e.g., electroporation, lipofection), enzymatic reactions, purificationand related techniques and procedures may be generally performed asdescribed in various general and more specific references inmicrobiology, molecular biology, biochemistry, molecular genetics, cellbiology, virology and immunology as cited and discussed throughout thepresent specification. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Current Protocols in Molecular Biology (John Wileyand Sons, updated July 2008); Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: APractical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA,1985); Current Protocols in Immunology (Edited by: John E. Coligan, AdaM. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology andApplications, Edited by Julie Logan, Kirstin Edwards and Nick Saunders,2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for theAnalysis of Complex Genomes, (Academic Press, New York, 1992); Guthrieand Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press,New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); NucleicAcid The Hybridization (B. Hames & S. Higgins, Eds., 1985);Transcription and Translation (B. Hames & S. Higgins, Eds., 1984);Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz,2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park,Ed., 3rd Edition, 2010 Humana Press); Immobilized Cells And Enzymes (IRLPress, 1986); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane,Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayerand Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and CC Blackwell,eds., 1986); Roitt, Essential Immunology, 6th Edition, (BlackwellScientific Publications, Oxford, 1988); Current Protocols in Immunology(Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W.Strober, eds., 1991); Annual Review of Immunology; as well as monographsin journals such as Advances in Immunology.

B. Definitions

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of particular embodiments, preferred embodimentsof compositions, methods and materials are described herein. For thepurposes of the present disclosure, the following terms are definedbelow.

The articles “a,” “an,” and “the” are used herein to refer to one or tomore than one (i.e., to at least one, or to one or more) of thegrammatical object of the article. By way of example, “an element” meansone element or one or more elements.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives.

The term “and/or” should be understood to mean either one, or both ofthe alternatives.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range ofquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%,±2%, or ±1% about a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phraseand limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are present that materially affect the activity or action ofthe listed elements.

Reference throughout this specification to “one embodiment,” “anembodiment,” “a particular embodiment,” “a related embodiment,” “acertain embodiment,” “an additional embodiment,” or “a furtherembodiment” or combinations thereof means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments. It is also understoodthat the positive recitation of a feature in one embodiment, serves as abasis for excluding the feature in a particular embodiment.

An “antigen (Ag)” refers to a compound, composition, or substance thatcan stimulate the production of antibodies or a T cell response in ananimal, including compositions (such as one that includes acancer-specific protein) that are injected or absorbed into an animal.Exemplary antigens include but are not limited to lipids, carbohydrates,polysaccharides, glycoproteins, peptides, or nucleic acids. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous antigens, such as the disclosedantigens.

A “target antigen” or “target antigen of interest” is an antigen that abinding domain contemplated herein, is designed to bind. In particularembodiments, the target antigen is selected from the group consistingof: alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX,CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD44, CD44v6, CD44v7/8, CD70,CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR familyincluding ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP,fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1,HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1,HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin,Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1,SSX, Survivin, STn, TAG72, TEMs, VEGFR2, and WT-1. In one embodiment,the antigen is an MHC-peptide complex, such as a class I MHC-peptidecomplex or a class II MHC-peptide complex.

An “antibody” refers to a binding agent that is a polypeptide comprisingat least a light chain or heavy chain immunoglobulin variable regionwhich specifically recognizes and binds an epitope of an antigen, suchas a lipid, carbohydrate, polysaccharide, glycoprotein, peptide, ornucleic acid containing an antigenic determinant, such as thoserecognized by an immune cell.

An “epitope” or “antigenic determinant” refers to the region of anantigen to which a binding agent binds.

Antibodies include antigen binding fragments thereof, such as a CamelIg, a Llama Ig, an Alpaca Ig, Ig NAR, a Fab′ fragment, a F(ab′)₂fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer(Fab3), an Fv, an single chain Fv protein (“scFv”), a bis-scFv, (scFv)₂,a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilizedFv protein (“dsFv”), and a single-domain antibody (sdAb, a camelid VHH,Nanobody) and portions of full length antibodies responsible for antigenbinding. The term also includes genetically engineered forms such aschimeric antibodies (for example, humanized murine antibodies),heteroconjugate antibodies (such as, bispecific antibodies) and antigenbinding fragments thereof. See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology,3rd Ed., W. H. Freeman & Co., New York, 1997.

A “linker” refers to a plurality of amino acid residues between thevarious polypeptide domains added for appropriate spacing andconformation of the molecule. In particular embodiments, the linker is avariable region linking sequence. A “variable region linking sequence,”is an amino acid sequence that connects the V_(H) and V_(L) domains andprovides a spacer function compatible with interaction of the twosub-binding domains so that the resulting polypeptide retains a specificbinding affinity to the same target molecule as an antibody thatcomprises the same light and heavy chain variable regions. In particularembodiments, a linker separates one or more heavy or light chainvariable domains, hinge domains, multimerization domains, transmembranedomains, co-stimulatory domains, and/or primary signaling domains.

Illustrated examples of linkers suitable for use in particularembodiments contemplated herein include, but are not limited to thefollowing amino acid sequences: GGG; DGGGS (SEQ ID NO: 7); TGEKP (SEQ IDNO: 8) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO:9) (Pomerantz et al. 1995, supra); (GGGGS)n wherein n=1, 2, 3, 4 or 5(SEQ ID NO: 10) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD(SEQ ID NO: 11) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A.87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 12) (Bird et al., 1988,Science 242:423-426), GGRRGGGS (SEQ ID NO: 13); LRQRDGERP (SEQ ID NO:14); LRQKDGGGSERP (SEQ ID NO: 15); LRQKD(GGGS)₂ ERP (SEQ ID NO: 16).Alternatively, flexible linkers can be rationally designed using acomputer program capable of modeling both DNA-binding sites and thepeptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS91:11099-11103 (1994) or by phage display methods. In one embodiment,the linker comprises the following amino acid sequence:GSTSGSGKPGSGEGSTKG (SEQ ID NO: 17) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

A “spacer domain,” refers to a polypeptide that separates two domains.In one embodiment, a spacer domain moves an antigen binding domain awayfrom the effector cell surface to enable proper cell/cell contact,antigen binding and activation (Patel et al., Gene Therapy, 1999; 6:412-419). In particular embodiments, a spacer domain separates one ormore heavy or light chain variable domains, multimerization domains,transmembrane domains, co-stimulatory domains, and/or primary signalingdomains. The spacer domain may be derived either from a natural,synthetic, semi-synthetic, or recombinant source. In certainembodiments, a spacer domain is a portion of an immunoglobulin,including, but not limited to, one or more heavy chain constant regions,e.g., CH2 and CH3. The spacer domain can include the amino acid sequenceof a naturally occurring immunoglobulin hinge region or an alteredimmunoglobulin hinge region.

A “hinge domain,” refers to a polypeptide that plays a role inpositioning the antigen binding domain away from the effector cellsurface to enable proper cell/cell contact, antigen binding andactivation. In particular embodiments, polypeptides may comprise one ormore hinge domains between the binding domain and the multimerizationdomain, between the binding domain and the transmembrane domain (TM), orbetween the multimerization domain and the transmembrane domain. Thehinge domain may be derived either from a natural, synthetic,semi-synthetic, or recombinant source. The hinge domain can include theamino acid sequence of a naturally occurring immunoglobulin hinge regionor an altered immunoglobulin hinge region.

A “multimerization domain,” as used herein, refers to a polypeptide thatpreferentially interacts or associates with another differentpolypeptide directly or via a bridging molecule, e.g., a chemicallyinducible dimerizer, wherein the interaction of differentmultimerization domains substantially contributes to or efficientlypromotes multimerization (i.e., the formation of a dimer, trimer, ormultipartite complex, which may be a homodimer, heterodimer, homotrimer,heterotrimer, homomultimer, heteromultimer). A multimerization domainmay be derived either from a natural, synthetic, semi-synthetic, orrecombinant source.

Illustrative examples of multimerization domains suitable for use inparticular embodiments contemplated herein include an FK506 bindingprotein (FKBP) polypeptide or variants thereof, an FKBP12-rapamycinbinding (FRB) polypeptide or variants thereof, a calcineurin polypeptideor variants thereof, a cyclophilin polypeptide or variants thereof, abacterial dihydrofolate reductase (DHFR) polypeptide or variantsthereof, a PYR1-like 1 (PYL1) polypeptide or variants thereof, anabscisic acid insensitive 1 (ABI1) polypeptide or variants thereof, aGIB1 polypeptide or variants thereof, or a GAI polypeptide or variantsthereof.

As used herein, the term “FKBP-rapamycin binding polypeptide” refers toan FRB polypeptide. In particular embodiments, the FRB polypeptide is anFKBP12-rapamycin binding polypeptide. FRB polypeptides suitable for usein particular embodiments contemplated herein generally contain at leastabout 85 to about 100 amino acid residues. In certain embodiments, theFRB polypeptide comprises a 93 amino acid sequence Ile-2021 throughLys-2113 and a mutation of T2098L, with reference to GenBank AccessionNo. L34075.1. An FRB polypeptide contemplated herein binds to an FKBPpolypeptide through a bridging factor, thereby forming a tripartitecomplex.

As used herein, the term “FK506 binding protein” refers to an FKBPpolypeptide. In particular embodiments, the FKBP polypeptide is anFKBP12 polypeptide. In certain embodiments, an FKBP domain may also bereferred to as a “rapamycin binding domain”. Information concerning thenucleotide sequences, cloning, and other aspects of various FKBP speciesis known in the art (see, e.g., Staendart et al., Nature 346:671, 1990(human FKBP12); Kay, Biochem. J. 314:361, 1996). An FKBP polypeptidecontemplated herein binds to an FRB polypeptide through a bridgingfactor, thereby forming a tripartite complex.

A “bridging factor” refers to a molecule that associates with and thatis disposed between two or more multimerization domains. In particularembodiments, multimerization domains substantially contribute to orefficiently promote formation of a polypeptide complex only in thepresence of a bridging factor. In particular embodiments,multimerization domains do not contribute to or do not efficientlypromote formation of a polypeptide complex in the absence of a bridgingfactor. Illustrative examples of bridging factors suitable for use inparticular embodiments contemplated herein include, but are not limitedto AP21967, rapamycin (sirolimus) or a rapalog thereof, coumermycin or aderivative thereof, gibberellin or a derivative thereof, abscisic acid(ABA) or a derivative thereof, methotrexate or a derivative thereof,cyclosporin A or a derivative thereof, FKCsA or a derivative thereof,trimethoprim (Tmp)-synthetic ligand for FKBP (SLF) or a derivativethereof, or any combination thereof.

Rapamycin analogs (rapalogs) include but are not limited to thosedisclosed in U.S. Pat. No. 6,649,595, which rapalog structures areincorporated herein by reference in their entirety. In certainembodiments, a bridging factor is a rapalog with substantially reducedimmunosuppressive effect as compared to rapamycin. In a preferredembodiment, the rapalog is AP21967 (also known asC-16-(S)-7-methylindolerapamycin, IC₅₀=10 nM, a chemically modifiednon-immunosuppressive rapamycin analogue). Other illustrative rapalogssuitable for use in particular embodiments contemplated herein include,but are not limited to, everolimus, novolimus, pimecrolimus,ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

A “substantially reduced immunosuppressive effect” refers to at leastless than 0.1 to 0.005 times the immunosuppressive effect observed orexpected for the same dose measured either clinically or in anappropriate in vitro (e.g., inhibition of T cell proliferation) or invivo surrogate of human immunosuppressive activity.

A “transmembrane domain” or “TM domain” is a domain that anchors apolypeptide to the plasma membrane of a cell. The TM domain may bederived either from a natural, synthetic, semi-synthetic, or recombinantsource.

An “intracellular signaling domain” or “endodomain” refers to theportion of a protein which transduces the effector function signal andthat directs the cell to perform a specialized function. While usuallythe entire intracellular signaling domain can be employed, in many casesit is not necessary to use the entire domain. To the extent that atruncated portion of an intracellular signaling domain is used, suchtruncated portion may be used in place of the entire domain as long asit transduces the effector function signal. The term intracellularsignaling domain is meant to include any truncated portion of theintracellular signaling domain sufficient to transducing effectorfunction signal.

The term “effector function” or “effector cell function” refers to aspecialized function of an immune effector cell. Effector functionincludes, but is not limited to, activation, cytokine production,proliferation and cytotoxic activity, including the release of cytotoxicfactors, or other cellular responses elicited with antigen binding tothe receptor expressed on the immune effector cell.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of intracellular signalingdomains: primary signaling domains that initiate antigen-dependentprimary activation through the TCR (e.g., a TCR/CD3 complex) andco-stimulatory signaling domains that act in an antigen-independentmanner to provide a secondary or co-stimulatory signal.

A “primary signaling domain” refers to a signaling domain that regulatesthe primary activation of the TCR complex either in a stimulatory way,or in an inhibitory way. Primary signaling domains that act in astimulatory manner may contain signaling motifs which are known asimmunoreceptor tyrosine-based activation motifs or ITAMs. Illustrativeexamples of ITAM containing primary signaling domains that are suitablefor use in particular embodiments include, but are not limited to thosederived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, andCD66d.

As used herein, the term, “co-stimulatory signaling domain,” or“co-stimulatory domain” refers to an intracellular signaling domain of aco-stimulatory molecule. Co-stimulatory molecules are cell surfacemolecules other than antigen receptors or Fc receptors that provide asecond signal required for efficient activation and function of Tlymphocytes upon binding to antigen. Illustrative examples of suchco-stimulatory molecules from which co-stimulatory domains may beisolated include, but are not limited to: TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40,CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10,LAT, NKD2C, SLP76, TRIM, and ZAP70.

An “immune disorder” refers to a disease that evokes a response from theimmune system. In particular embodiments, the term “immune disorder”refers to a cancer, an autoimmune disease, or an immunodeficiency. Inone embodiment, immune disorders encompass infectious disease.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues.

As used herein, the term “malignant” refers to a cancer in which a groupof tumor cells display one or more of uncontrolled growth (i.e.,division beyond normal limits), invasion (i.e., intrusion on anddestruction of adjacent tissues), and metastasis (i.e., spread to otherlocations in the body via lymph or blood). As used herein, the term“metastasize” refers to the spread of cancer from one part of the bodyto another. A tumor formed by cells that have spread is called a“metastatic tumor” or a “metastasis.” The metastatic tumor containscells that are like those in the original (primary) tumor.

As used herein, the term “benign” or “non-malignant” refers to tumorsthat may grow larger but do not spread to other parts of the body.Benign tumors are self-limited and typically do not invade ormetastasize.

A “cancer cell” refers to an individual cell of a cancerous growth ortissue. Cancer cells include both solid cancers and liquid cancers. A“tumor” or “tumor cell” refers generally to a swelling or lesion formedby an abnormal growth of cells, which may be benign, pre-malignant, ormalignant. Most cancers form tumors, but liquid cancers, e.g., leukemia,do not necessarily form tumors. For those cancers that form tumors, theterms cancer (cell) and tumor (cell) are used interchangeably. Theamount of a tumor in an individual is the “tumor burden” which can bemeasured as the number, volume, or weight of the tumor.

The term “relapse” refers to the diagnosis of return, or signs andsymptoms of return, of a cancer after a period of improvement orremission.

“Remission,” is also referred to as “clinical remission,” and includesboth partial and complete remission. In partial remission, some, but notall, signs and symptoms of cancer have disappeared. In completeremission, all signs and symptoms of cancer have disappeared, althoughcancer still may be in the body.

“Refractory” refers to a cancer that is resistant to, or non-responsiveto, therapy with a particular therapeutic agent. A cancer can berefractory from the onset of treatment (i.e., non-responsive to initialexposure to the therapeutic agent), or as a result of developingresistance to the therapeutic agent, either over the course of a firsttreatment period or during a subsequent treatment period.

“Antigen negative” refers to a cell that does not express antigen orexpresses a negligible amount of antigen that is undetectable. In oneembodiment, antigen negative cells do not bind receptors directed to theantigen. In one embodiment, antigen negative cells do not substantiallybind receptors directed to the antigen.

An “autoimmune disease” refers to a disease in which the body producesan immunogenic (i.e., immune system) response to some constituent of itsown tissue. In other words, the immune system loses its ability torecognize some tissue or system within the body as “self” and targetsand attacks it as if it were foreign. Autoimmune diseases can beclassified into those in which predominantly one organ is affected(e.g., hemolytic anemia and anti-immune thyroiditis), and those in whichthe autoimmune disease process is diffused through many tissues (e.g.,systemic lupus erythematosus). For example, multiple sclerosis isthought to be caused by T cells attacking the sheaths that surround thenerve fibers of the brain and spinal cord. This results in loss ofcoordination, weakness, and blurred vision. Autoimmune diseases areknown in the art and include, for instance, Hashimoto's thyroiditis,Grave's disease, lupus, multiple sclerosis, rheumatic arthritis,hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,celiac disease, Crohn's disease, colitis, diabetes, scleroderma,psoriasis, and the like.

An “immunodeficiency” means the state of a patient whose immune systemhas been compromised by disease or by administration of chemicals. Thiscondition makes the system deficient in the number and type of bloodcells needed to defend against a foreign substance. Immunodeficiencyconditions or diseases are known in the art and include, for example,AIDS (acquired immunodeficiency syndrome), SCID (severe combinedimmunodeficiency disease), selective IgA deficiency, common variableimmunodeficiency, X-linked agammaglobulinemia, chronic granulomatousdisease, hyper-IgM syndrome, and diabetes.

An “infectious disease” refers to a disease that can be transmitted fromperson to person or from organism to organism and is caused by amicrobial or viral agent (e.g., common cold). Infectious diseases areknown in the art and include, for example, hepatitis, sexuallytransmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis,HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.

As used herein, the terms “individual” and “subject” are often usedinterchangeably and refer to any animal that exhibits a symptom ofcancer or other immune disorder that can be treated with thecompositions and methods contemplated elsewhere herein. Suitablesubjects (e.g., patients) include laboratory animals (such as mouse,rat, rabbit, or guinea pig), farm animals, and domestic animals or pets(such as a cat or dog). Non-human primates and, preferably, humanpatients, are included. Typical subjects include human patients thathave, have been diagnosed with, or are at risk or having, cancer oranother immune disorder.

As used herein, the term “patient” refers to a subject that has beendiagnosed with cancer or another immune disorder that can be treatedwith the compositions and methods disclosed elsewhere herein.

As used herein “treatment” or “treating,” includes any beneficial ordesirable effect on the symptoms or pathology of a disease orpathological condition and may include even minimal reductions in one ormore measurable markers of the disease or condition being treated.Treatment can involve optionally either the reduction of the disease orcondition, or the delaying of the progression of the disease orcondition, e.g., delaying tumor outgrowth. “Treatment” does notnecessarily indicate complete eradication or cure of the disease orcondition, or associated symptoms thereof.

As used herein, “prevent,” and similar words such as “prevented,”“preventing” etc., indicate an approach for preventing, inhibiting, orreducing the likelihood of the occurrence or recurrence of, a disease orcondition. It also refers to delaying the onset or recurrence of adisease or condition or delaying the occurrence or recurrence of thesymptoms of a disease or condition. As used herein, “prevention” andsimilar words also includes reducing the intensity, effect, symptomsand/or burden of a disease or condition prior to onset or recurrence ofthe disease or condition.

As used herein, the phrase “ameliorating at least one symptom of” refersto decreasing one or more symptoms of the disease or condition for whichthe subject is being treated. In particular embodiments, the disease orcondition being treated is a cancer, wherein the one or more symptomsameliorated include, but are not limited to, weakness, fatigue,shortness of breath, easy bruising and bleeding, frequent infections,enlarged lymph nodes, distended or painful abdomen (due to enlargedabdominal organs), bone or joint pain, fractures, unplanned weight loss,poor appetite, night sweats, persistent mild fever, and decreasedurination (due to impaired kidney function).

By “enhance” or “promote,” or “increase” or “expand” refers generally tothe ability of a composition contemplated herein to produce, elicit, orcause a greater physiological response (i.e., downstream effects)compared to the response caused by either vehicle or a controlmolecule/composition. A measurable physiological response may include anincrease in T cell expansion, activation, persistence, cytokinesecretion, and/or an increase in cancer cell killing ability, amongothers apparent from the understanding in the art and the descriptionherein. An “increased” or “enhanced” amount is typically a“statistically significant” amount, and may include an increase that is1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the responseproduced by vehicle or a control composition.

By “decrease” or “lower,” or “lessen,” or “reduce,” or “abate” refersgenerally to the ability of composition contemplated herein to produce,elicit, or cause a lesser physiological response (i.e., downstreameffects) compared to the response caused by either vehicle or a controlmolecule/composition. A “decrease” or “reduced” amount is typically a“statistically significant” amount, and may include a decrease that is1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times(e.g., 500, 1000 times) (including all integers and decimal points inbetween and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response(reference response) produced by vehicle, a control composition, or theresponse in a particular cell lineage.

By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “nosubstantial change,” or “no substantial decrease” refers generally tothe ability of a composition contemplated herein to produce, elicit, orcause a substantially similar or comparable physiological response(i.e., downstream effects) in a cell, as compared to the response causedby either vehicle, a control molecule/composition, or the response in aparticular cell lineage. A comparable response is one that is notsignificantly different or measurable different from the referenceresponse.

Additional definitions are set forth throughout this disclosure.

C. DARIC Immune Receptors

In particular embodiments, one or more DARIC immune receptors thattransduce an immunostimulatory signal upon exposure to a bridging factoris contemplated. As used herein, the term “DARIC immune receptor” refersto one or more non-naturally occurring polypeptides that transduces animmunostimulatory signal in an immune effector cell upon exposure to amultimerizing agent or bridging factor, e.g., stimulating immuneeffector cell activity and function, increasing production and/orsecretion of proinflammatory cytokines.

In particular embodiments, the DARIC immune receptor is a polypeptidecomprising a first multimerization domain, a transmembrane domain, anintracellular signaling domain of an immune receptor, e.g., a humanimmune receptor, including, but not limited to a cytokine receptor, aninterleukin receptor, a pattern recognition receptor, and a toll-likereceptor; a polypeptide cleavage signal; and a second multimerizationdomain, a transmembrane domain, and an intracellular signaling domain ofan immune receptor, e.g., a human immune receptor, including, but notlimited to a cytokine receptor, an interleukin receptor, a patternrecognition receptor, and a toll-like receptor.

In other particular embodiments, the DARIC immune receptor is a complexof polypeptides comprising a first polypeptide comprising a firstmultimerization domain, a transmembrane domain, and an intracellularsignaling domain of an immune receptor including, but not limited to acytokine receptor, an interleukin receptor, a pattern recognitionreceptor, and a toll-like receptor; and a second polypeptide comprisinga second multimerization domain, a transmembrane domain, and anintracellular signaling domain of an immune receptor including, but notlimited to a cytokine receptor, an interleukin receptor, a patternrecognition receptor, and a toll-like receptor.

As used herein, the term “immune receptor” refers to a receptor that isexpressed on the surface of an immune cell that modulates an immuneresponse upon binding its cognate ligand. Immune receptors suitable foruse in particular embodiments include, but are not limited to: cytokinereceptors, interleukin receptors, pattern recognition receptors, andtoll-like receptors, wherein signaling through the immune receptorstimulates an immune response.

Illustrative examples of multimerization domains suitable for use inparticular DARIC immune receptors contemplated herein include, but arenot limited to, an FK506 binding protein (FKBP) polypeptide or variantsthereof, an FKBP12-rapamycin binding (FRB) polypeptide or variantsthereof, a calcineurin polypeptide or variants thereof, a cyclophilinpolypeptide or variants thereof, a bacterial dihydrofolate reductase(DHFR) polypeptide or variants thereof, a PYR1-like 1 (PYL1) polypeptideor variants thereof, an abscisic acid insensitive 1 (ABI1) polypeptideor variants thereof, a GIB1 polypeptide or variants thereof, or a GAIpolypeptide or variants thereof.

Illustrative examples of immune receptor transmembrane domains suitablefor use in particular DARIC immune receptors contemplated hereininclude, but are not limited to, the transmembrane region(s) of thealpha, beta, gamma, or delta chain of a T-cell receptor, CD3ε, CD3ζ,CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64,CD71, CD80, CD86, CD 134, CD137, CD152, CD 154, AMN, PD1, IL-12Rβ2,IL-7Rα, IL-2Rγ, IL-2Rβ, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP,IFNAR1, IFNAR2, IL-1RL2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, or TLR10.

Illustrative examples of immune receptor intracellular signaling domainssuitable for use in particular DARIC immune receptors contemplatedherein include, but are not limited to, intracellular signaling domainsisolated from an IL-12 receptor, an IL-7 receptor, an IL-15 receptor, anIL-21 receptor, an IL-2 receptor, an IL-1 receptor, an IL-18 receptor,an IL-36 receptor, a type I IFN receptor, a TLR1 receptor, a TLR2receptor, a TLR3 receptor, a TLR4 receptor, a TLR5 receptor, a TLR6receptor, a TLR7 receptor, a TLR8 receptor, a TLR9 receptor, or a TLR10receptor.

Illustrative examples of immune receptor intracellular signaling domainssuitable for use in particular DARIC immune receptors contemplatedherein include, but are not limited to, IL-12Rβ2, IL-7Rα, IL-2Rγ,IL-2R13, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1, IFNAR2,IL-1RL2, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10.

Illustrative examples of cytokine receptor intracellular signalingdomains suitable for use in particular DARIC immune receptorscontemplated herein include, but are not limited to, IL-12Rβ2, IL-7Rα,IL-2Rγ, IL-2Rβ, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, IFNAR1,IFNAR2, and IL-1RL2.

Illustrative examples of interleukin receptor intracellular signalingdomains suitable for use in particular DARIC immune receptorscontemplated herein include, but are not limited to, IL-12Rβ2, IL-7Rα,IL-2Rγ, IL-2Rβ, IL-21R, IL-18R1, IL-18RAP, IL-1R1, IL-1RAP, and IL-1RL2.

Illustrative examples of toll-like receptor intracellular signalingdomains suitable for use in particular DARIC immune receptorscontemplated herein include, but are not limited to, TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

1. IL-18 DARIC Immune Receptor

Interleukin-18 (IL-18) is a cytokine that promotes T cell function andactivity by, in part, increasing IFNγ expression, increasing T cellproliferation, and protecting against activation induced cell death(AICD). IL-18 binds interleukin 18 receptor 1, (IL-18R1, also known asCD218a) and interleukin 18 receptor accessory protein (IL-18RAP,CD218b).

IL-18 signaling through IL-18R1 and IL-18RAP results in activationthrough the MyD88 adaptor protein and IRAK4 phosphorylation.Phosphorylation of IRAK4 and subsequent phosphorylation of IRAK1/2ultimately leads to activation of NF-kappa B and AP-1 transcriptionfactors to increase IFNγ expression and increase sensitivity to IL-12.The transcriptional program induced by IL-18 also increases T cellproliferation and protects against AICD.

In various embodiments, one or more immune effector cells, includingimmune effector cells expressing an engineered antigen receptor, aremodified by introducing one or more polynucleotides or vectors encodingan IL-18 DARIC immune receptor. In various embodiments, one or moreimmune effector cells are modified by introducing one or morepolynucleotides or vectors encoding an IL-18 DARIC immune receptor andan engineered antigen receptor.

In particular embodiments, the IL-18 DARIC immune receptor transduces anIL-18-mediated immunostimulatory signal upon exposure to a bridgingfactor. In particular embodiments an IL-18 DARIC immune receptorcontemplated herein comprises: a first multimerization domain, atransmembrane domain, and an IL-18RAP intracellular signaling domain; apolypeptide cleavage signal; and a second multimerization domain, atransmembrane domain, and an IL-18R1 intracellular signaling domain. Inparticular embodiments an IL-18 DARIC immune receptor contemplatedherein comprises: a first multimerization domain, a transmembranedomain, and an IL-18R1 intracellular signaling domain; a polypeptidecleavage signal; and a second multimerization domain, a transmembranedomain, and an IL-18RAP intracellular signaling domain.

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide; more preferably, a viral self-cleaving 2Apolypeptide; and more preferably a viral self-cleaving polypeptideselected from the group consisting of: a foot-and-mouth disease virus(FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, aThosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1)(P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditisvirus 2A peptide. In one embodiment, the polypeptide cleavage signal isa P2A or T2A viral self-cleaving polypeptide.

In particular embodiments, the IL-18 DARIC immune receptor is a complexof polypeptides comprising a first polypeptide comprising a firstmultimerization domain, a first transmembrane domain, and an IL-18RAPintracellular signaling domain; and a polypeptide comprising a secondmultimerization domain, a second transmembrane domain, and an IL-18R1intracellular signaling domain. In particular embodiments, an IL-18immune receptor is a complex of polypeptides comprising a firstpolypeptide comprising a first multimerization domain, a firsttransmembrane domain, and an IL-18R1 intracellular signaling domain; anda polypeptide comprising a second multimerization domain, a secondtransmembrane domain, and an IL-18RAP intracellular signaling domain.

In particular embodiments, the first and second transmembrane domainsare selected from the group consisting of: CD4, CD5, CD8α, CD9, CD 16,CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134,CD137, CD152, CD 154, AMN, and PD1.

In one embodiment, the first and second multimerization domains are thesame.

In certain embodiments, an IL-18 DARIC immune receptor comprises a firstor second multimerization domain selected from a pair selected from FKBPand FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterialDHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, orvariants thereof.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8atransmembrane domain, and an IL-18R1 intracellular signaling domain; anda second polypeptide comprises a multimerization domain comprising anFRB polypeptide or variant thereof, a CD4 or CD8a transmembrane domain,and an IL-18RAP intracellular signaling domain.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FRB polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-18R1 intracellular signaling domain; anda second polypeptide comprises a multimerization domain comprising anFKBP polypeptide or variant thereof, a CD4 or CD8α transmembrane domain,and an IL-18RAP intracellular signaling domain.

In some embodiments, the bridging factor is AP21967, sirolimus,everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,temsirolimus, umirolimus, or zotarolimus.

In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBPpolypeptide is FKBP12; and the bridging factor is AP21967.

In particular embodiments, the FRB polypeptide is FRB; the FKBPpolypeptide is FKBP12; and the bridging factor is rapamycin,temsirolimus or everolimus.

2. IL-12 DARIC Immune Receptor

Interleukin-12 (IL-12) is a cytokine that promotes T cell function andactivity by, in part, increasing IFNγ expression, increasing T cellproliferation, and potentiating IL-12 signaling. IL-12 binds interleukin12 receptor, beta 1 (IL-12Rβ1, also known as CD212) and interleukin 12receptor, beta 2 (IL-12Rβ2).

IL-12 signaling through IL-12Rβ1 and IL-12Rβ2 results in STAT3, STAT4,and STAT5 phosphorylation. Phosphorylated STAT3/STAT4 translocates tothe nucleus and binds the IFNγ promoter to increase IFNγ expression.Phosphorylated STAT4 also recruits Jun oncogene (c-Jun) to IFNγ promoterto increase IFNγ expression and potentiates IL-12 signaling byincreasing transcription of IL-12Rβ2. STAT5 phosphorylation increases Tcell proliferation.

IL-12 signaling also increases expression of interleukin 2 receptor,alpha (IL-2R) by recruiting STAT4 and c-Jun to the promoter of IL-2R,thereby enhancing T cell proliferation.

In various embodiments, one or more immune effector cells, includingimmune effector cells expressing an engineered antigen receptor, aremodified by introducing one or more polynucleotides or vectors encodingan IL-12 DARIC immune receptor. In various embodiments, one or moreimmune effector cells are modified by introducing one or morepolynucleotides or vectors encoding an IL-12 DARIC immune receptor andan engineered antigen receptor.

In particular embodiments, the IL-12 DARIC immune receptor transduces anIL-12-mediated immunostimulatory signal upon exposure to a bridgingfactor. In particular embodiments an IL-12 DARIC immune receptorcontemplated herein comprises: a first multimerization domain, atransmembrane domain, and an IL-12Rβ1 intracellular signaling domain; apolypeptide cleavage signal; and a second multimerization domain, atransmembrane domain, and an IL-12Rβ2 intracellular signaling domain. Inparticular embodiments an IL-12 DARIC immune receptor contemplatedherein comprises: a first multimerization domain, a transmembranedomain, and an IL-12Rβ2 intracellular signaling domain; a polypeptidecleavage signal; and a second multimerization domain, a transmembranedomain, and an IL-12Rβ1 intracellular signaling domain.

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide; more preferably, a viral self-cleaving 2Apolypeptide; and more preferably a viral self-cleaving polypeptideselected from the group consisting of: a foot-and-mouth disease virus(FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, aThosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1)(P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditisvirus 2A peptide. In one embodiment, the polypeptide cleavage signal isa P2A or T2A viral self-cleaving polypeptide.

In particular embodiments, the IL-12 DARIC immune receptor is a complexof polypeptides comprising a first polypeptide comprising a firstmultimerization domain, a first transmembrane domain, and an IL-12Rβ1intracellular signaling domain; and a polypeptide comprising a secondmultimerization domain, a second transmembrane domain, and an IL-12Rβ2intracellular signaling domain. In particular embodiments, an IL-12immune receptor is a complex of polypeptides comprising a firstpolypeptide comprising a first multimerization domain, a firsttransmembrane domain, and an IL-12Rβ2 intracellular signaling domain;and a polypeptide comprising a second multimerization domain, a secondtransmembrane domain, and an IL-12Rβ1 intracellular signaling domain.

In particular embodiments, the first and second transmembrane domainsare selected from the group consisting of: CD4, CD5, CD8α, CD9, CD 16,CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134,CD137, CD152, CD 154, AMN, and PD1.

In one embodiment, the first and second multimerization domains are thesame.

In certain embodiments, an IL-12 DARIC immune receptor comprises a firstor second multimerization domain selected from a pair selected from FKBPand FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterialDHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, orvariants thereof.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-12Rβ1 intracellular signaling domain;and a second polypeptide comprises a multimerization domain comprisingan FRB polypeptide or variant thereof, a CD4 or CD8α transmembranedomain, and an IL-12Rβ2 intracellular signaling domain.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FRB polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-12Rβ1 intracellular signaling domain;and a second polypeptide comprises a multimerization domain comprisingan FKBP polypeptide or variant thereof, a CD4 or CD8α transmembranedomain, and an IL-12Rβ2 intracellular signaling domain.

In some embodiments, the bridging factor is AP21967, sirolimus,everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,temsirolimus, umirolimus, or zotarolimus.

In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBPpolypeptide is FKBP12; and the bridging factor is AP21967.

In particular embodiments, the FRB polypeptide is FRB; the FKBPpolypeptide is FKBP12; and the bridging factor is rapamycin,temsirolimus or everolimus.

3. IL-7 DARIC Immune Receptor

Interleukin-7 (IL-7) is a cytokine that promotes T cell function andactivity by, in part, improving T cell precursor survival andproliferation. IL-7 binds interleukin 7 receptor alpha (IL-7Rα, alsoknown as CD127) and interleukin 2 receptor, common gamma chain (IL-2Rγ,also known as CD132 and γc). IL-7 signaling activates the JAK/STAT,PI-3K, and Src kinase pathways and results in transcription ofanti-apoptotic genes and genes that promote proliferation of T cellprecursors.

In various embodiments, one or more immune effector cells, includingimmune effector cells expressing an engineered antigen receptor, aremodified by introducing one or more polynucleotides or vectors encodingan IL-7 DARIC immune receptor. In various embodiments, one or moreimmune effector cells are modified by introducing one or morepolynucleotides or vectors encoding an IL-7 DARIC immune receptor and anengineered antigen receptor.

In particular embodiments, the IL-7 DARIC immune receptor transduces anIL-7-mediated immunostimulatory signal upon exposure to a bridgingfactor. In particular embodiments an IL-7 DARIC immune receptorcontemplated herein comprises: a first multimerization domain, atransmembrane domain, and an IL-7Rα intracellular signaling domain; apolypeptide cleavage signal; and a second multimerization domain, atransmembrane domain, and an IL-2Rγ intracellular signaling domain. Inparticular embodiments an IL-7 DARIC immune receptor contemplated hereincomprises: a first multimerization domain, a transmembrane domain, andan IL-2Rγ intracellular signaling domain; a polypeptide cleavage signal;and a second multimerization domain, a transmembrane domain, and anIL-7Rα intracellular signaling domain.

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide; more preferably, a viral self-cleaving 2Apolypeptide; and more preferably a viral self-cleaving polypeptideselected from the group consisting of: a foot-and-mouth disease virus(FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, aThosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1)(P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditisvirus 2A peptide. In one embodiment, the polypeptide cleavage signal isa P2A or T2A viral self-cleaving polypeptide.

In particular embodiments, the IL-7 DARIC immune receptor is a complexof polypeptides comprising a first polypeptide comprising a firstmultimerization domain, a first transmembrane domain, and an IL-7Rαintracellular signaling domain; and a polypeptide comprising a secondmultimerization domain, a second transmembrane domain, and an IL-2Rγintracellular signaling domain. In particular embodiments, an IL-7immune receptor is a complex of polypeptides comprising a firstpolypeptide comprising a first multimerization domain, a firsttransmembrane domain, and an IL-2Rγ intracellular signaling domain; anda polypeptide comprising a second multimerization domain, a secondtransmembrane domain, and an IL-7Rα intracellular signaling domain.

In particular embodiments, the first and second transmembrane domainsare selected from the group consisting of: CD4, CD5, CD8α, CD9, CD 16,CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134,CD137, CD152, CD 154, AMN, and PD1.

In one embodiment, the first and second multimerization domains are thesame.

In certain embodiments, an IL-7 DARIC immune receptor comprises a firstor second multimerization domain selected from a pair selected from FKBPand FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterialDHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, orvariants thereof.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-7Rα intracellular signaling domain; anda second polypeptide comprises a multimerization domain comprising anFRB polypeptide or variant thereof, a CD4 or CD8α transmembrane domain,and an IL-2Rγ intracellular signaling domain.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FRB polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-7Rαintracellular signaling domain; and asecond polypeptide comprises a multimerization domain comprising an FKBPpolypeptide or variant thereof, a CD4 or CD8α transmembrane domain, andan IL-2Rγ intracellular signaling domain.

In some embodiments, the bridging factor is AP21967, sirolimus,everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,temsirolimus, umirolimus, or zotarolimus.

In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBPpolypeptide is FKBP12; and the bridging factor is AP21967.

In particular embodiments, the FRB polypeptide is FRB; the FKBPpolypeptide is FKBP12; and the bridging factor is rapamycin,temsirolimus or everolimus.

4. IL-15 DARIC Immune Receptor

Interleukin-15 (IL-15) is a cytokine that promotes T cell function andactivity by, in part, improving T cell precursor survival andproliferation. IL-15 binds with high affinity to IL-15Rα (also known asCD215), which then associates with a complex comprising IL-2Rβ (alsoknown as IL-15Rβ and CD122) and IL-2Rγ (also known as CD132 and γc),expressed either on the same cell (cis-presentation) or on a differentcell (trans-presentation). IL-15 signaling activates the JAK/STAT,PI-3K, and Src kinase pathways and results in transcription ofanti-apoptotic genes and genes that promote proliferation of T cellprecursors.

In various embodiments, one or more immune effector cells, includingimmune effector cells expressing an engineered antigen receptor, aremodified by introducing one or more polynucleotides or vectors encodingan IL-15 DARIC immune receptor, and optionally, a polynucleotide orvector encoding an IL-15Rα polypeptide. In various embodiments, one ormore immune effector cells are modified by introducing one or morepolynucleotides or vectors encoding an IL-15 DARIC immune receptor andan engineered antigen receptor, and optionally, a polynucleotide orvector encoding an IL-15Rα polypeptide.

In particular embodiments, the IL-15 DARIC immune receptor transduces anIL-15-mediated immunostimulatory signal upon exposure to a bridgingfactor. In particular embodiments an IL-15 DARIC immune receptorcontemplated herein comprises: a first multimerization domain, atransmembrane domain, and an IL-2Rβ intracellular signaling domain; apolypeptide cleavage signal; and a second multimerization domain, atransmembrane domain, and an IL-2Rγ intracellular signaling domain. Inparticular embodiments an IL-15 DARIC immune receptor contemplatedherein comprises: a first multimerization domain, a transmembranedomain, and an IL-2Rγ intracellular signaling domain; a polypeptidecleavage signal; and a second multimerization domain, a transmembranedomain, and an IL-2Rβ intracellular signaling domain.

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide; more preferably, a viral self-cleaving 2Apolypeptide; and more preferably a viral self-cleaving polypeptideselected from the group consisting of: a foot-and-mouth disease virus(FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, aThosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1)(P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditisvirus 2A peptide. In one embodiment, the polypeptide cleavage signal isa P2A or T2A viral self-cleaving polypeptide.

In particular embodiments, the IL-15 DARIC immune receptor is a complexof polypeptides comprising a first polypeptide comprising a firstmultimerization domain, a first transmembrane domain, and an IL-2Rβintracellular signaling domain; and a polypeptide comprising a secondmultimerization domain, a second transmembrane domain, and an IL-2Rγintracellular signaling domain. In particular embodiments, an IL-15immune receptor is a complex of polypeptides comprising a firstpolypeptide comprising a first multimerization domain, a firsttransmembrane domain, and an IL-2Rγ intracellular signaling domain; anda polypeptide comprising a second multimerization domain, a secondtransmembrane domain, and an IL-2Rβ intracellular signaling domain.

In particular embodiments, the first and second transmembrane domainsare selected from the group consisting of: CD4, CD5, CD8α, CD9, CD 16,CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134,CD137, CD152, CD 154, AMN, and PD1.

In one embodiment, the first and second multimerization domains are thesame.

In certain embodiments, an IL-15 DARIC immune receptor comprises a firstor second multimerization domain selected from a pair selected from FKBPand FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterialDHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, orvariants thereof.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-2Rβ intracellular signaling domain; anda second polypeptide comprises a multimerization domain comprising anFRB polypeptide or variant thereof, a CD4 or CD8α transmembrane domain,and an IL-2Rγ intracellular signaling domain.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FRB polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-2Rβ intracellular signaling domain; anda second polypeptide comprises a multimerization domain comprising anFKBP polypeptide or variant thereof, a CD4 or CD8α transmembrane domain,and an IL-2Rγ intracellular signaling domain.

In some embodiments, the bridging factor is AP21967, sirolimus,everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,temsirolimus, umirolimus, or zotarolimus.

In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBPpolypeptide is FKBP12; and the bridging factor is AP21967.

In particular embodiments, the FRB polypeptide is FRB; the FKBPpolypeptide is FKBP12; and the bridging factor is rapamycin,temsirolimus or everolimus.

5. IL-21 DARIC Immune Receptor

Interleukin-21 (IL-21) is a cytokine that promotes T cell function andactivity by, in part, improving T cell precursor survival andproliferation. IL-21 binds to interleukin 21 receptor (IL-21R, alsoknown as CD360) and IL-2Rγ (also known as CD132 and γc). IL-21 signalingactivates the JAK/STAT, PI-3K, and Src kinase pathways and results intranscription of anti-apoptotic genes and genes that promoteproliferation of T cell precursors.

In various embodiments, one or more immune effector cells, includingimmune effector cells expressing an engineered antigen receptor, aremodified by introducing one or more polynucleotides or vectors encodingan IL-21 DARIC immune receptor. In various embodiments, one or moreimmune effector cells are modified by introducing one or morepolynucleotides or vectors encoding an IL-21 DARIC immune receptor andan engineered antigen receptor.

In particular embodiments, the IL-21 DARIC immune receptor transduces anIL-21-mediated immunostimulatory signal upon exposure to a bridgingfactor. In particular embodiments an IL-21 DARIC immune receptorcontemplated herein comprises: a first multimerization domain, atransmembrane domain, and an IL-21R intracellular signaling domain; apolypeptide cleavage signal; and a second multimerization domain, atransmembrane domain, and an IL-2Rγ intracellular signaling domain. Inparticular embodiments an IL-21 DARIC immune receptor contemplatedherein comprises: a first multimerization domain, a transmembranedomain, and an IL-2Rγ intracellular signaling domain; a polypeptidecleavage signal; and a second multimerization domain, a transmembranedomain, and an IL-21R intracellular signaling domain.

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide; more preferably, a viral self-cleaving 2Apolypeptide; and more preferably a viral self-cleaving polypeptideselected from the group consisting of: a foot-and-mouth disease virus(FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, aThosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1)(P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditisvirus 2A peptide. In one embodiment, the polypeptide cleavage signal isa P2A or T2A viral self-cleaving polypeptide.

In particular embodiments, the IL-21 DARIC immune receptor is a complexof polypeptides comprising a first polypeptide comprising a firstmultimerization domain, a first transmembrane domain, and an IL-21Rintracellular signaling domain; and a polypeptide comprising a secondmultimerization domain, a second transmembrane domain, and an IL-2Rγintracellular signaling domain. In particular embodiments, an IL-21immune receptor is a complex of polypeptides comprising a firstpolypeptide comprising a first multimerization domain, a firsttransmembrane domain, and an IL-2Rγ intracellular signaling domain; anda polypeptide comprising a second multimerization domain, a secondtransmembrane domain, and an IL-21R intracellular signaling domain.

In particular embodiments, the first and second transmembrane domainsare selected from the group consisting of: CD4, CD5, CD8α, CD9, CD 16,CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134,CD137, CD152, CD 154, AMN, and PD1.

In one embodiment, the first and second multimerization domains are thesame.

In certain embodiments, an IL-21 DARIC immune receptor comprises a firstor second multimerization domain selected from a pair selected from FKBPand FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterialDHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, orvariants thereof.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-21R intracellular signaling domain; anda second polypeptide comprises a multimerization domain comprising anFRB polypeptide or variant thereof, a CD4 or CD8α transmembrane domain,and an IL-2Rγ intracellular signaling domain.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FRB polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-21R intracellular signaling domain; anda second polypeptide comprises a multimerization domain comprising anFKBP polypeptide or variant thereof, a CD4 or CD8α transmembrane domain,and an IL-12Rγ intracellular signaling domain.

In some embodiments, the bridging factor is AP21967, sirolimus,everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,temsirolimus, umirolimus, or zotarolimus.

In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBPpolypeptide is FKBP12; and the bridging factor is AP21967.

In particular embodiments, the FRB polypeptide is FRB; the FKBPpolypeptide is FKBP12; and the bridging factor is rapamycin,temsirolimus or everolimus.

6. IL-1 DARIC Immune Receptor

Interleukin-1 (IL-1) is a cytokine that promotes T cell function andactivity by, in part, increasing IFNγ expression, increasing T cellproliferation, and potentiating protecting against activation inducedcell death (AICD). IL-1 binds interleukin 1 receptor 1, (IL-1R1, alsoknown as CD121a) and interleukin 1 receptor accessory protein (IL-1RAP).

IL-1 signaling through IL-1R1 and IL-TRAP results in activation throughthe MyD88 adaptor protein and IRAK4 phosphorylation. Phosphorylation ofIRAK4 and subsequent phosphorylation of IRAK1/2 ultimately leads toactivation of NF-kappa B and AP-1 transcription factors to increase IFNγexpression and increase sensitivity to IL-12. The transcriptionalprogram induced by IL-1 also increases T cell proliferation and protectsagainst AICD.

In various embodiments, one or more immune effector cells, includingimmune effector cells expressing an engineered antigen receptor, aremodified by introducing one or more polynucleotides or vectors encodingan IL-1 DARIC immune receptor. In various embodiments, one or moreimmune effector cells are modified by introducing one or morepolynucleotides or vectors encoding an IL-1 DARIC immune receptor and anengineered antigen receptor.

In particular embodiments, the IL-1 DARIC immune receptor transduces anIL-1-mediated immunostimulatory signal upon exposure to a bridgingfactor. In particular embodiments an IL-1 DARIC immune receptorcontemplated herein comprises: a first multimerization domain, atransmembrane domain, and an IL-1RAP intracellular signaling domain; apolypeptide cleavage signal; and a second multimerization domain, atransmembrane domain, and an IL-1R1 intracellular signaling domain. Inparticular embodiments an IL-1 DARIC immune receptor contemplated hereincomprises: a first multimerization domain, a transmembrane domain, andan IL-1R1 intracellular signaling domain; a polypeptide cleavage signal;and a second multimerization domain, a transmembrane domain, and anIL-1RAP intracellular signaling domain.

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide; more preferably, a viral self-cleaving 2Apolypeptide; and more preferably a viral self-cleaving polypeptideselected from the group consisting of: a foot-and-mouth disease virus(FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, aThosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1)(P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditisvirus 2A peptide. In one embodiment, the polypeptide cleavage signal isa P2A or T2A viral self-cleaving polypeptide.

In particular embodiments, the IL-1 DARIC immune receptor is a complexof polypeptides comprising a first polypeptide comprising a firstmultimerization domain, a first transmembrane domain, and an IL-1RAPintracellular signaling domain; and a polypeptide comprising a secondmultimerization domain, a second transmembrane domain, and an IL-1R1intracellular signaling domain. In particular embodiments, an IL-1immune receptor is a complex of polypeptides comprising a firstpolypeptide comprising a first multimerization domain, a firsttransmembrane domain, and an IL-1R1 intracellular signaling domain; anda polypeptide comprising a second multimerization domain, a secondtransmembrane domain, and an IL-1RAP intracellular signaling domain.

In particular embodiments, the first and second transmembrane domainsare selected from the group consisting of: CD4, CD5, CD8α, CD9, CD 16,CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134,CD137, CD152, CD 154, AMN, and PD1.

In one embodiment, the first and second multimerization domains are thesame.

In certain embodiments, an IL-1 DARIC immune receptor comprises a firstor second multimerization domain selected from a pair selected from FKBPand FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterialDHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, orvariants thereof.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-1RAP intracellular signaling domain; anda second polypeptide comprises a multimerization domain comprising anFRB polypeptide or variant thereof, a CD4 or CD8α transmembrane domain,and an IL-1R1 intracellular signaling domain.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FRB polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and an IL-1RAP intracellular signaling domain; anda second polypeptide comprises a multimerization domain comprising anFKBP polypeptide or variant thereof, a CD4 or CD8α transmembrane domain,and an IL-1R1 intracellular signaling domain.

In some embodiments, the bridging factor is AP21967, sirolimus,everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,temsirolimus, umirolimus, or zotarolimus.

In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBPpolypeptide is FKBP12; and the bridging factor is AP21967.

In particular embodiments, the FRB polypeptide is FRB; the FKBPpolypeptide is FKBP12; and the bridging factor is rapamycin,temsirolimus or everolimus.

7. TLR DARIC Immune Receptor

Toll like receptors (TLR1 through TLR10) are pattern recognitionreceptors that detect invading pathogens and activate the innate andadaptive immune responses. Activation of TLRs by various ligands leadsto induction of a pro-inflammatory transcriptional program andexpression of multiple inflammatory cytokines.

TLR signaling occurs via homodimerization of TLR signaling domainsleading to activation through the MyD88 adaptor protein and IRAK4phosphorylation. Phosphorylation of IRAK4 and subsequent phosphorylationof IRAK1/2 ultimately leads to activation of NF-kappa B and AP-1transcription factors to increase inflammatory cytokine production andinduce proliferation. TLR activation can also lead to the activation ofIRF3 and IRF7 transcription factors.

In various embodiments, one or more immune effector cells, includingimmune effector cells expressing an engineered antigen receptor, aremodified by introducing one or more polynucleotides or vectors encodinga TLR DARIC immune receptor. In various embodiments, one or more immuneeffector cells are modified by introducing one or more polynucleotidesor vectors encoding a TLR DARIC immune receptor and an engineeredantigen receptor.

In particular embodiments, the TLR DARIC immune receptor transduces aTLR mediated immunostimulatory signal upon exposure to a bridgingfactor. In particular embodiments TLR DARIC immune receptor contemplatedherein comprises: a first multimerization domain, a transmembranedomain, and a TLR intracellular signaling domain; a polypeptide cleavagesignal; and a second multimerization domain, a transmembrane domain, andan identical TLR intracellular signaling domain. In particularembodiments a TLR DARIC immune receptor contemplated herein comprises: afirst multimerization domain, a transmembrane domain, and a TLRintracellular signaling domain; a polypeptide cleavage signal; and asecond multimerization domain, a transmembrane domain, and an identicalTLR intracellular signaling domain.

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving polypeptide; more preferably, a viral self-cleaving 2Apolypeptide; and more preferably a viral self-cleaving polypeptideselected from the group consisting of: a foot-and-mouth disease virus(FMDV) (F2A) peptide, an equine rhinitis A virus (ERAV) (E2A) peptide, aThosea asigna virus (TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1)(P2A) peptide, a Theilovirus 2A peptide, and an encephalomyocarditisvirus 2A peptide. In one embodiment, the polypeptide cleavage signal isa P2A or T2A viral self-cleaving polypeptide.

In particular embodiments, the TLR DARIC immune receptor is a complex ofpolypeptides comprising a first polypeptide comprising a firstmultimerization domain, a first transmembrane domain, and a TLRintracellular signaling domain; and a polypeptide comprising a secondmultimerization domain, a second transmembrane domain, and an identicalTLR intracellular signaling domain. In particular embodiments, a TLRimmune receptor is a complex of polypeptides comprising a firstpolypeptide comprising a first multimerization domain, a firsttransmembrane domain, and a TLR intracellular signaling domain; and apolypeptide comprising a second multimerization domain, a secondtransmembrane domain, and an identical TLR intracellular signalingdomain.

In preferred embodiments, the TLR intracellular signaling domain isisolated from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, orTLR10.

In particular embodiments, the first and second transmembrane domainsare selected from the group consisting of: CD4, CD5, CD8α, CD9, CD 16,CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD71, CD80, CD86, CD 134,CD137, CD152, CD 154, AMN, and PD1.

In one embodiment, the first and second multimerization domains are thesame.

In certain embodiments, a TLR DARIC immune receptor comprises a first orsecond multimerization domain selected from a pair selected from FKBPand FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterialDHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, orvariants thereof.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FKBP polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and a TLR intracellular signaling domain; and asecond polypeptide comprises a multimerization domain comprising an FRBpolypeptide or variant thereof, a CD4 or CD8α transmembrane domain, andan identical TLR intracellular signaling domain.

In certain embodiments, a first polypeptide comprises a multimerizationdomain comprising an FRB polypeptide or variant thereof, a CD4 or CD8αtransmembrane domain, and a TLR intracellular signaling domain; and asecond polypeptide comprises a multimerization domain comprising an FKBPpolypeptide or variant thereof, a CD4 or CD8α transmembrane domain, andan identical TLR intracellular signaling domain.

In some embodiments, the bridging factor is AP21967, sirolimus,everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus,temsirolimus, umirolimus, or zotarolimus.

In particular embodiments, the FRB polypeptide is FRB T2098L; the FKBPpolypeptide is FKBP12; and the bridging factor is AP21967.

In particular embodiments, the FRB polypeptide is FRB; the FKBPpolypeptide is FKBP12; and the bridging factor is rapamycin,temsirolimus or everolimus.

D. Engineered Antigen Receptors

In particular embodiments, a polypeptide comprises an engineered antigenreceptor, a polypeptide cleavage signal and a DARIC immune receptor. Inother particular embodiments, a polynucleotide or vector encoding aDARIC immune receptor is introduced into an immune effector cell thatcomprises an engineered antigen receptor. Without wishing to be bound byany particular theory, it is contemplated in particular embodiments,that any mechanism known in the art may be used to introduce andco-express an engineered antigen receptor and a DARIC immune receptor inthe same immune effector cell or population of cells to increase theresistance of the immune effector cells to the TME and potentiate andincrease the efficiency, potency, and durability of the immune effectorcell response.

In particular embodiments, immune effector cells contemplated hereincomprise an engineered antigen receptor and a DARIC immune receptor. Inparticular embodiments, the engineered antigen receptor is an engineeredT cell receptor (TCR), a chimeric antigen receptor (CAR), or a zetakine.

1. Engineered TCRs

In particular embodiments, immune effector cells contemplated hereincomprise an engineered TCR and a DARIC immune receptor. In oneembodiment, T cells are engineered by introducing a polynucleotide orvector encoding an engineered TCR and a DARIC immune receptor separatedby one or more polypeptide cleavage signals. In one embodiment, T cellsare engineered by introducing a polynucleotide or vector encoding anengineered TCR and a polynucleotide or vector encoding a DARIC immunereceptor. In one embodiment, T cells are engineered to express anengineered TCR are further engineered by introducing a polynucleotide orvector encoding a DARIC immune receptor.

Naturally occurring T cell receptors comprise two subunits, an alphachain and a beta chain subunit, each of which is a unique proteinproduced by recombination event in each T cell's genome. Libraries ofTCRs may be screened for their selectivity to particular targetantigens. In this manner, natural TCRs, which have a high-avidity andreactivity toward target antigens may be selected, cloned, andsubsequently introduced into a population of T cells used for adoptiveimmunotherapy.

In one embodiment, T cells are modified by introducing a TCR subunit hasthe ability to form TCRs that confer specificity to T cells for tumorcells expressing a target antigen. In particular embodiments, thesubunits have one or more amino acid substitutions, deletions,insertions, or modifications compared to the naturally occurringsubunit, so long as the subunits retain the ability to form TCRs andconfer upon transfected T cells the ability to home to target cells andparticipate in immunologically-relevant cytokine signaling. Theengineered TCRs preferably also bind target cells displaying therelevant tumor-associated peptide with high avidity, and optionallymediate efficient killing of target cells presenting the relevantpeptide in vivo.

The nucleic acids encoding engineered TCRs are preferably isolated fromtheir natural context in a (naturally-occurring) chromosome of a T celland can be incorporated into suitable vectors as described elsewhereherein. Both the nucleic acids and the vectors comprising them can betransferred into a cell, preferably a T cell in particular embodiments.The modified T cells are then able to express one or more chains of aTCR encoded by the transduced nucleic acid or nucleic acids. Inpreferred embodiments, the engineered TCR is an exogenous TCR because itis introduced into T cells that do not normally express the particularTCR. The essential aspect of the engineered TCRs is that it has highavidity for a tumor antigen presented by a major histocompatibilitycomplex (MHC) or similar immunological component. In contrast toengineered TCRs, CARs are engineered to bind target antigens in an MHCindependent manner.

The TCR can be expressed with additional polypeptides attached to theamino-terminal or carboxyl-terminal portion of the alpha chain or betachain of a TCR so long as the attached additional polypeptide does notinterfere with the ability of the alpha chain or beta chain to form afunctional T cell receptor and the MHC dependent antigen recognition.

Antigens that are recognized by the engineered TCRs contemplated inparticular embodiments include, but are not limited to cancer antigens,including antigens on both hematological cancers and solid tumors.Illustrative antigens include, but are not limited to alpha folatereceptor, alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6,CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a,CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family includingErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetalAchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1,HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1,IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16,NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin,TAG72, TEMs, VEGFR2, and WT-1.

2. Chimeric Antigen Receptors

In various embodiments, immune effector cells express CARs that redirectcytotoxicity toward tumor cells. CARs are molecules that combineantibody-based specificity for a target antigen (e.g., tumor antigen)with a T cell receptor-activating intracellular domain to generate achimeric protein that exhibits a specific anti-tumor cellular immuneactivity. As used herein, the term, “chimeric,” describes being composedof parts of different proteins or DNAs from different origins.

In particular embodiments, immune effector cells contemplated hereincomprise CAR and a DARIC immune receptor. In one embodiment, T cells areengineered by introducing a polynucleotide or vector encoding a CAR anda DARIC immune receptor separated by one or more polypeptide cleavagesignals. In one embodiment, T cells are engineered by introducing apolynucleotide or vector encoding a CAR and a polynucleotide or vectorencoding a DARIC immune receptor. In one embodiment, T cells areengineered to express a CAR are further engineered by introducing apolynucleotide or vector encoding a DARIC immune receptor.

In various embodiments, a CAR comprises an extracellular domain thatbinds to a specific target antigen (also referred to as a binding domainor antigen-specific binding domain), a transmembrane domain and anintracellular signaling domain. The main characteristic of CARs is theirability to redirect immune effector cell specificity, thereby triggeringproliferation, cytokine production, phagocytosis or production ofmolecules that can mediate cell death of the target antigen expressingcell in a major histocompatibility (WIC) independent manner, exploitingthe cell specific targeting abilities of monoclonal antibodies, solubleligands or cell specific coreceptors.

In particular embodiments, CARs comprise an extracellular binding domainthat specifically binds to a target polypeptide. A binding domainincludes any naturally occurring, synthetic, semi-synthetic, orrecombinantly produced binding partner for a biological molecule ofinterest.

In particular embodiments, the extracellular binding domain comprises anantibody or antigen binding fragment thereof.

In one preferred embodiment, the binding domain is an scFv.

In another preferred embodiment, the binding domain is a camelidantibody.

In particular embodiments, the CAR comprises an extracellular domainthat binds an antigen selected from the group consisting of: alphafolate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16,CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a,CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family includingErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetalAchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1,HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1,IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16,NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin,TAG72, TEMs, VEGFR2, and WT-1.

In particular embodiments, the CARs comprise an extracellular bindingdomain, e.g., antibody or antigen binding fragment thereof that binds anantigen, wherein the antigen is an MHC-peptide complex, such as a classI MHC-peptide complex or a class II MHC-peptide complex.

In one embodiment, the spacer domain comprises the CH2 and CH3 of IgG1,IgG4, or IgD.

Illustrative hinge domains suitable for use in the CARs described hereininclude the hinge region derived from the extracellular regions of type1 membrane proteins such as CD8α, and CD4, which may be wild-type hingeregions from these molecules or may be altered. In another embodiment,the hinge domain comprises a CD8α hinge region.

In one embodiment, the hinge is a PD-1 hinge or CD152 hinge.

The transmembrane domain (TM) of the CAR fuses the extracellular bindingportion and intracellular signaling domain and anchors the CAR to theplasma membrane of the immune effector cell. The TM domain may bederived either from a natural, synthetic, semi-synthetic, or recombinantsource.

Illustrative TM domains may be derived from (i.e., comprise at least thetransmembrane region(s) of the alpha, beta, gamma, or delta chain of theT-cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16,CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137,CD152, CD154, AMN, and PD-1.

In one embodiment, a CAR comprises a TM domain derived from CD8α. Inanother embodiment, a CAR contemplated herein comprises a TM domainderived from CD8α and a short oligo- or polypeptide linker, preferablybetween 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length thatlinks the TM domain and the intracellular signaling domain of the CAR. Aglycine-serine linker provides a particularly suitable linker.

In preferred embodiments, a CAR comprises an intracellular signalingdomain that comprises one or more “co-stimulatory signaling domains” anda “primary signaling domain.”

Primary signaling domains that act in a stimulatory manner may containsignaling motifs which are known as immunoreceptor tyrosine-basedactivation motifs or ITAMs.

Illustrative examples of ITAM containing primary signaling domainssuitable for use in CARs contemplated in particular embodiments includethose derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a,CD79b, and CD66d. In particular preferred embodiments, a CAR comprises aCD3ζ primary signaling domain and one or more co-stimulatory signalingdomains. The intracellular primary signaling and co-stimulatorysignaling domains may be linked in any order in tandem to the carboxylterminus of the transmembrane domain.

In particular embodiments, a CAR comprises one or more co-stimulatorysignaling domains to enhance the efficacy and expansion of T cellsexpressing CAR receptors.

Illustrative examples of such co-stimulatory molecules suitable for usein CARs contemplated in particular embodiments include TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28,CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278(ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In one embodiment, aCAR comprises one or more co-stimulatory signaling domains selected fromthe group consisting of CD28, CD137, and CD134, and a CD3ζ primarysignaling domain.

In various embodiments, the CAR comprises: an extracellular domain thatbinds an antigen selected from the group consisting of: BCMA, CD19,CSPG4, PSCA, ROR1, and TAG72; a transmembrane domain isolated from apolypeptide selected from the group consisting of: CD4, CD8α, CD154, andPD-1; one or more intracellular co-stimulatory signaling domainsisolated from a polypeptide selected from the group consisting of: CD28,CD134, and CD137; and a signaling domain isolated from a polypeptideselected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε,CD3ζ, CD22, CD79a, CD79b, and CD66d.

3. Zetakines

In various embodiments, immune effector cells comprise chimeric cytokinereceptor that redirect cytotoxicity toward tumor cells. Zetakines arechimeric transmembrane immunoreceptors that comprise an extracellulardomain comprising a soluble receptor ligand linked to a support regioncapable of tethering the extracellular domain to a cell surface, atransmembrane region and an intracellular signaling domain. Zetakines,when expressed on the surface of T lymphocytes, direct T cell activityto those cells expressing a receptor for which the soluble receptorligand is specific. Zetakine chimeric immunoreceptors redirect theantigen specificity of T cells, with application to treatment of avariety of cancers, particularly via the autocrine/paracrine cytokinesystems utilized by human malignancy.

In particular embodiments, immune effector cells contemplated hereincomprise one or more chains of a zetakine receptor and a DARIC immunereceptor. In one embodiment, T cells are engineered by introducing apolynucleotide or vector encoding one or more chains of a zetakinereceptor and a DARIC immune receptor separated by one or morepolypeptide cleavage signals. In one embodiment, T cells are engineeredby introducing a polynucleotide or vector encoding one or more chains ofa zetakine receptor and a polynucleotide or vector encoding a DARICimmune receptor. In one embodiment, T cells are engineered to expressone or more chains of a zetakine receptor are further engineered byintroducing a polynucleotide or vector encoding a DARIC immune receptor.

In particular embodiments, the zetakine comprises an immunosuppressivecytokine or cytokine receptor binding variant thereof, a linker, atransmembrane domain, and an intracellular signaling domain.

In particular embodiments, the cytokine or cytokine receptor bindingvariant thereof is selected from the group consisting of: interleukin-4(IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10(IL-10), and interleukin-13 (IL-13).

In certain embodiments, the linker comprises a CH2CH3 domain, hingedomain, or the like. In one embodiment, a linker comprises the CH2 andCH3 domains of IgG1, IgG4, or IgD. In one embodiment, a linker comprisesa CD8α or CD4 hinge domain.

In particular embodiments, the transmembrane domain is selected from thegroup consisting of: the alpha or beta chain of the T-cell receptor,CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28,CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN,and PD-1.

In particular embodiments, the intracellular signaling domain isselected from the group consisting of: an ITAM containing primarysignaling domain and/or a co-stimulatory domain.

In particular embodiments, the intracellular signaling domain isselected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε,CD3′, CD22, CD79a, CD79b, and CD66d.

In particular embodiments, the intracellular signaling domain isselected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40,CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10,LAT, NKD2C, SLP76, TRIM, and ZAP70.

In one embodiment, a chimeric cytokine receptor comprises one or moreco-stimulatory signaling domains selected from the group consisting ofCD28, CD137, and CD134, and a CD3ζ primary signaling domain.

E. Polypeptides

Various polypeptides are contemplated herein, including, but not limitedto, DARIC immune receptors, engineered TCRs, CARs, zetakines, fusionproteins comprising the foregoing polypeptides and fragments thereof. Inpreferred embodiments, a polypeptide comprises an amino acid sequenceset forth in any one of SEQ ID NOs: 1-6. “Polypeptide,” “peptide” and“protein” are used interchangeably, unless specified to the contrary,and according to conventional meaning, i.e., as a sequence of aminoacids. In one embodiment, a “polypeptide” includes fusion polypeptidesand other variants. Polypeptides can be prepared using any of a varietyof well-known recombinant and/or synthetic techniques. Polypeptides arenot limited to a specific length, e.g., they may comprise a full-lengthprotein sequence, a fragment of a full-length protein, or a fusionprotein, and may include post-translational modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring. In particular preferredembodiments, fusion polypeptides, polypeptides, fragments and othervariants thereof are prepared, obtained, or isolated from one or morehuman polypeptides.

An “isolated peptide” or an “isolated polypeptide” and the like, as usedherein, refer to in vitro isolation and/or purification of a peptide orpolypeptide molecule from a cellular environment, and from associationwith other components of the cell, i.e., it is not significantlyassociated with in vivo substances. In particular embodiments, anisolated polypeptide is a synthetic polypeptide, a semi-syntheticpolypeptide, or a polypeptide obtained or derived from a recombinantsource.

Polypeptides include “polypeptide variants.” Polypeptide variants maydiffer from a naturally occurring polypeptide in one or moresubstitutions, deletions, additions and/or insertions. Such variants maybe naturally occurring or may be synthetically generated, for example,by modifying one or more of the above polypeptide sequences. Forexample, in particular embodiments, it may be desirable to improve thebinding affinity and/or other biological properties of a polypeptide byintroducing one or more substitutions, deletions, additions and/orinsertions the polypeptide. In particular embodiments, polypeptidesinclude polypeptides having at least about 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or99% amino acid identity to any of the reference sequences contemplatedherein, typically where the variant maintains at least one biologicalactivity of the reference sequence. In particular embodiments, thebiological activity is binding affinity. In particular embodiments, thebiological activity is enzymatic activity.

In certain embodiments, a polypeptide complex comprises (i) a firstpolypeptide, e.g., first fusion polypeptide, having a firstmultimerization domain and (ii) second polypeptide, e.g., second fusionpolypeptide, having a second multimerization domain that is not the sameas the first multimerization domain, wherein the first and secondmultimerization domains substantially contribute to or efficientlypromote formation of the polypeptide complex in the presence of abridging factor. The interaction(s) between the first and secondmultimerization domains substantially contributes to or efficientlypromotes the multimerization of the first and second fusion polypeptidesif there is a statistically significant reduction in the associationbetween the first and second fusion polypeptides in the absence of thefirst multimerization domain, the second multimerization domain, or thebridging factor. In certain embodiments, when the first and secondfusion polypeptides are co-expressed, at least about 60%, for instance,at least about 60% to about 70%, at least about 70% to about 80%, atleast about 80% to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%, and at least about 90% to about 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% of the first and second single chain polypeptides formmultimers with each other in the presence of a bridging factor.

Polypeptides variants include biologically active “polypeptidefragments.” Illustrative examples of biologically active polypeptidefragments include binding domains, signaling domains, and the like. Asused herein, the term “biologically active fragment” or “minimalbiologically active fragment” refers to a polypeptide fragment thatretains at least 100%, at least 90%, at least 80%, at least 70%, atleast 60%, at least 50%, at least 40%, at least 30%, at least 20%, atleast 10%, or at least 5% of the naturally occurring polypeptideactivity. In certain embodiments, a polypeptide fragment can comprise anamino acid chain at least 5 to about 1700 amino acids long. It will beappreciated that in certain embodiments, fragments are at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 ormore amino acids long.

In particular embodiments, the polypeptides set forth herein maycomprise one or more amino acids denoted as “X.” “X” if present in anamino acid SEQ ID NO, refers to any one or more amino acids. Inparticular embodiments, SEQ ID NOs denoting a fusion protein comprise asequence of continuous X residues that cumulatively represent any scFv.

As noted above, polypeptides may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a reference polypeptide can beprepared by mutations in the DNA. Methods for mutagenesis and nucleotidesequence alterations are well known in the art. See, for example, Kunkel(1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987,Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J.D. et al., (Molecular Biology of the Gene, Fourth Edition,Benjamin/Cummings, Menlo Park, Calif., 1987) and the references citedtherein. Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al., (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found, Washington, D.C.).

In certain embodiments, a polypeptide variant comprises one or moreconservative substitutions. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged. Modifications may be madein the structure of the polynucleotides and polypeptides contemplated inparticular embodiments and still obtain a functional molecule thatencodes a variant or derivative polypeptide with desirablecharacteristics. When it is desired to alter the amino acid sequence ofa polypeptide to create an equivalent, or even an improved, variantpolypeptide, one skilled in the art, for example, can change one or moreof the codons of the encoding DNA sequence, e.g., according to Table 1.

TABLE 1 Amino Acid Codons One Three Amino letter letter Acids code codeCodons Alanine A Ala GCA GCC GCG GCU Cysteine C Cys UGC UGU Asparticacid D Asp GAC GAU Glutamic acid E Glu GAA GAG Phenylalanine F Phe UUCUUU Glycine G Gly GGA GGC GGG GGU Histidine H His CAC CAU Isoleucine IIso AUA AUC AUU Lysine K Lys AAA AAG Leucine L Leu UUA UUG CUA CUC CUGCUU Methionine M Met AUG Asparagine N Asn AAC AAU Proline P Pro CCA CCCCCG CCU Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGUSerine S Ser AGC AGU UCA UCC UCG UCU Threonine T Thr ACA ACC ACG ACUValine V Val GUA GUC GUG GUU Tryptophan W Trp UGG Tyrosine Y Tyr UAC UAU

Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological activity can be foundusing computer programs well known in the art, such as DNASTAR, DNAStrider, Geneious, Mac Vector, or Vector NTI software. Preferably, aminoacid changes in the protein variants disclosed herein are conservativeamino acid changes, i.e., substitutions of similarly charged oruncharged amino acids. A conservative amino acid change involvessubstitution of one of a family of amino acids which are related intheir side chains. Naturally occurring amino acids are generally dividedinto four families: acidic (aspartate, glutamate), basic (lysine,arginine, histidine), non-polar (alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), and uncharged polar(glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine)amino acids. Phenylalanine, tryptophan, and tyrosine are sometimesclassified jointly as aromatic amino acids. In a peptide or protein,suitable conservative substitutions of amino acids are known to those ofskill in this art and generally can be made without altering abiological activity of a resulting molecule. Those of skill in this artrecognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson et al. Molecular Biology of theGene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p. 224).

In one embodiment, where expression of two or more polypeptides isdesired, the polynucleotide sequences encoding them can be separated byan IRES sequence as disclosed elsewhere herein.

Polypeptides contemplated in particular embodiments include fusionpolypeptides. In particular embodiments, fusion polypeptides andpolynucleotides encoding fusion polypeptides are provided. Fusionpolypeptides and fusion proteins refer to a polypeptide having at leasttwo, three, four, five, six, seven, eight, nine, or ten polypeptidesegments. In preferred embodiments, a fusion polypeptide is a DARICimmune receptor. In other preferred embodiments, the fusion polypeptidecomprises one or more DARIC immune receptors. In particular preferredembodiments, fusion polypeptides comprise one or more segments,fragments, or domains of one or more human polypeptides.

In another embodiment, two or more polypeptides can be expressed as afusion protein that comprises one or more self-cleaving polypeptidesequences as disclosed elsewhere herein.

Fusion polypeptides can comprise one or more polypeptide domains orsegments including, but are not limited to signal peptides, cellpermeable peptide domains (CPP), binding domains, signaling domains,etc., epitope tags (e.g., maltose binding protein (“MBP”), glutathione Stransferase (GST), HIS6, MYC, FLAG, V5, VSV-G, and HA), polypeptidelinkers, and polypeptide cleavage signals. Fusion polypeptides aretypically linked C-terminus to N-terminus, although they can also belinked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminusto C-terminus. In particular embodiments, the polypeptides of the fusionprotein can be in any order. Fusion polypeptides or fusion proteins canalso include conservatively modified variants, polymorphic variants,alleles, mutants, subsequences, and interspecies homologs, so long asthe desired activity of the fusion polypeptide is preserved. Fusionpolypeptides may be produced by chemical synthetic methods or bychemical linkage between the two moieties or may generally be preparedusing other standard techniques. Ligated DNA sequences comprising thefusion polypeptide are operably linked to suitable transcriptional ortranslational control elements as disclosed elsewhere herein.

Fusion polypeptides may optionally comprise a linker that can be used tolink the one or more polypeptides or domains within a polypeptide. Apeptide linker sequence may be employed to separate any two or morepolypeptide components by a distance sufficient to ensure that eachpolypeptide folds into its appropriate secondary and tertiary structuresso as to allow the polypeptide domains to exert their desired functions.Such a peptide linker sequence is incorporated into the fusionpolypeptide using standard techniques in the art. Suitable peptidelinker sequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. Linkersequences are not required when a particular fusion polypeptide segmentcontains non-essential N-terminal amino acid regions that can be used toseparate the functional domains and prevent steric interference.Preferred linkers are typically flexible amino acid subsequences whichare synthesized as part of a recombinant fusion protein. Linkerpolypeptides can be between 1 and 200 amino acids in length, between 1and 100 amino acids in length, or between 1 and 50 amino acids inlength, including all integer values in between.

Exemplary polypeptide cleavage signals include polypeptide cleavagerecognition sites such as protease cleavage sites, nuclease cleavagesites (e.g., rare restriction enzyme recognition sites, self-cleavingribozyme recognition sites), and self-cleaving viral oligopeptides (seedeFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

Suitable protease cleavages sites and self-cleaving peptides are knownto the skilled person (see, e.g., in Ryan et al., 1997. J. Gener. Virol.78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).Exemplary protease cleavage sites include, but are not limited to thecleavage sites of potyvirus NIa proteases (e.g., tobacco etch virusprotease), potyvirus HC proteases, potyvirus P1 (P35) proteases,byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus Lproteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3Cproteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (ricetungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleckvirus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.Due to its high cleavage stringency, TEV (tobacco etch virus) proteasecleavage sites are preferred in one embodiment, e.g., EXXYXQ(G/S) (SEQID NO: 18), for example, ENLYFQG (SEQ ID NO: 19) and ENLYFQS (SEQ ID NO:20), wherein X represents any amino acid (cleavage by TEV occurs betweenQ and G or Q and S).

In particular embodiments, the polypeptide cleavage signal is a viralself-cleaving peptide or ribosomal skipping sequence.

Illustrative examples of ribosomal skipping sequences include but arenot limited to: a 2A or 2A-like site, sequence or domain (Donnelly etal., 2001. J. Gen. Virol. 82:1027-1041). In a particular embodiment, theviral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide,or a cardiovirus 2A peptide.

In one embodiment, the viral 2A peptide is selected from the groupconsisting of: a foot-and-mouth disease virus (FMDV) (F2A) peptide, anequine rhinitis A virus (ERAV) (E2A) peptide, a Thosea asigna virus(TaV) (T2A) peptide, a porcine teschovirus-1 (PTV-1) (P2A) peptide, aTheilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

Illustrative examples of 2A sites are provided in Table 2.

TABLE 2 SEQ ID NO: 21 GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 22ATNFSLLKQAGDVEENPGP SEQ ID NO: 23 LLKQAGDVEENPGP SEQ ID NO: 24GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 25 EGRGSLLTCGDVEENPGP SEQ ID NO: 26LLTCGDVEENPGP SEQ ID NO: 27 GSGQCTNYALLKLAGDVESNPGP SEQ ID NO: 28QCTNYALLKLAGDVESNPGP SEQ ID NO: 29 LLKLAGDVESNPGP SEQ ID NO: 30GSGVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 31 VKQTLNFDLLKLAGDVESNPGPSEQ ID NO: 32 LLKLAGDVESNPGP SEQ ID NO: 33 LLNFDLLKLAGDVESNPGPSEQ ID NO: 34 TLNFDLLKLAGDVESNPGP SEQ ID NO: 35 LLKLAGDVESNPGPSEQ ID NO: 36 NFDLLKLAGDVESNPGP SEQ ID NO: 37 QLLNFDLLKLAGDVESNPGPSEQ ID NO: 38 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 39VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAP VKQT SEQ ID NO: 40LNFDLLKLAGDVESNPGP SEQ ID NO: 41 LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 42 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

In preferred embodiments, a polypeptide or fusion polypeptide comprisesa DARIC immune receptor polypeptide.

F. Polynucleotides

In particular embodiments, polynucleotides encoding DARIC immunereceptors, engineered TCRs, CARs, zetakines, fusion proteins comprisingthe foregoing polypeptides and fragments thereof are provided. As usedherein, the terms “polynucleotide” or “nucleic acid” refer todeoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA/RNA hybrids.Polynucleotides may be single-stranded or double-stranded and eitherrecombinant, synthetic, or isolated. Polynucleotides include, but arenot limited to: pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA,short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA(miRNA), ribozymes, genomic RNA (gRNA), plus strand RNA (RNA(+)), minusstrand RNA (RNA(−)), tracrRNA, crRNA, single guide RNA (sgRNA),synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA,complementary DNA (cDNA), synthetic DNA, or recombinant DNA.Polynucleotides refer to a polymeric form of nucleotides of at least 5,at least 10, at least 15, at least 20, at least 25, at least 30, atleast 40, at least 50, at least 100, at least 200, at least 300, atleast 400, at least 500, at least 1000, at least 5000, at least 10000,or at least 15000 or more nucleotides in length, either ribonucleotidesor deoxyribonucleotides or a modified form of either type of nucleotide,as well as all intermediate lengths. It will be readily understood that“intermediate lengths,” in this context, means any length between thequoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152,153, etc.; 201, 202, 203, etc. In particular embodiments,polynucleotides or variants have at least or about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to a reference sequence.

In particular embodiments, polynucleotides may be codon-optimized. Asused herein, the term “codon-optimized” refers to substituting codons ina polynucleotide encoding a polypeptide in order to increase theexpression, stability and/or activity of the polypeptide. Factors thatinfluence codon optimization include, but are not limited to one or moreof: (i) variation of codon biases between two or more organisms or genesor synthetically constructed bias tables, (ii) variation in the degreeof codon bias within an organism, gene, or set of genes, (iii)systematic variation of codons including context, (iv) variation ofcodons according to their decoding tRNAs, (v) variation of codonsaccording to GC %, either overall or in one position of the triplet,(vi) variation in degree of similarity to a reference sequence forexample a naturally occurring sequence, (vii) variation in the codonfrequency cutoff, (viii) structural properties of mRNAs transcribed fromthe DNA sequence, (ix) prior knowledge about the function of the DNAsequences upon which design of the codon substitution set is to bebased, (x) systematic variation of codon sets for each amino acid,and/or (xi) isolated removal of spurious translation initiation sites.

As used herein the term “nucleotide” refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a phosphorylated sugar.Nucleotides are understood to include natural bases, and a wide varietyof art-recognized modified bases. Such bases are generally located atthe 1′ position of a nucleotide sugar moiety. Nucleotides generallycomprise a base, sugar and a phosphate group. In ribonucleic acid (RNA),the sugar is a ribose, and in deoxyribonucleic acid (DNA) the sugar is adeoxyribose, i.e., a sugar lacking a hydroxyl group that is present inribose. Exemplary natural nitrogenous bases include the purines,adenosine (A) and guanidine (G), and the pyrimidines, cytidine (C) andthymidine (T) (or in the context of RNA, uracil (U)). The C-1 atom ofdeoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine.Nucleotides are usually mono, di- or triphosphates. The nucleotides canbe unmodified or modified at the sugar, phosphate and/or base moiety,(also referred to interchangeably as nucleotide analogs, nucleotidederivatives, modified nucleotides, non-natural nucleotides, andnon-standard nucleotides; see for example, WO 92/07065 and WO 93/15187).Examples of modified nucleic acid bases are summarized by Limbach etal., (1994, Nucleic Acids Res. 22, 2183-2196).

A nucleotide may also be regarded as a phosphate ester of a nucleoside,with esterification occurring on the hydroxyl group attached to C-5 ofthe sugar. As used herein, the term “nucleoside” refers to aheterocyclic nitrogenous base in N-glycosidic linkage with a sugar.Nucleosides are recognized in the art to include natural bases, and alsoto include well known modified bases. Such bases are generally locatedat the position of a nucleoside sugar moiety. Nucleosides generallycomprise a base and sugar group. The nucleosides can be unmodified ormodified at the sugar, and/or base moiety, (also referred tointerchangeably as nucleoside analogs, nucleoside derivatives, modifiednucleosides, non-natural nucleosides, or non-standard nucleosides). Asalso noted above, examples of modified nucleic acid bases are summarizedby Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).

Illustrative examples of polynucleotides include but are not limited topolynucleotides encoding SEQ ID NOs: 1-6.

In various illustrative embodiments, polynucleotides contemplated hereininclude, but are not limited to polynucleotides encoding DARIC immunereceptors, engineered antigen receptors, fusion polypeptides, andexpression vectors, viral vectors, and transfer plasmids comprisingpolynucleotides contemplated herein.

As used herein, the terms “polynucleotide variant” and “variant” and thelike refer to polynucleotides displaying substantial sequence identitywith a reference polynucleotide sequence or polynucleotides thathybridize with a reference sequence under stringent conditions that aredefined hereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletion,substitution, or modification of at least one nucleotide. Accordingly,the terms “polynucleotide variant” and “variant” include polynucleotidesin which one or more nucleotides have been added or deleted, ormodified, or replaced with different nucleotides. In this regard, it iswell understood in the art that certain alterations inclusive ofmutations, additions, deletions and substitutions can be made to areference polynucleotide whereby the altered polynucleotide retains thebiological function or activity of the reference polynucleotide.

In one embodiment, a polynucleotide comprises a nucleotide sequence thathybridizes to a target nucleic acid sequence under stringent conditions.To hybridize under “stringent conditions” describes hybridizationprotocols in which nucleotide sequences at least 60% identical to eachother remain hybridized. Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength, pH and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. Since thetarget sequences are generally present at excess, at Tm, 50% of theprobes are occupied at equilibrium.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Included are nucleotides and polypeptides having at leastabout 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequenceidentity to any of the reference sequences described herein, typicallywhere the polypeptide variant maintains at least one biological activityof the reference polypeptide.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity,” and “substantial identity”. A “reference sequence” is atleast 12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons Inc, 1994-1998, Chapter 15.

As used herein, “isolated polynucleotide” refers to a polynucleotidethat has been purified from the sequences which flank it in anaturally-occurring state, e.g., a DNA fragment that has been removedfrom the sequences that are normally adjacent to the fragment. An“isolated polynucleotide” also refers to a complementary DNA (cDNA), arecombinant DNA, or other polynucleotide that does not exist in natureand that has been made by the hand of man. In particular embodiments, anisolated polynucleotide is a synthetic polynucleotide, a semi-syntheticpolynucleotide, or a polynucleotide obtained or derived from arecombinant source.

In various embodiments, a polynucleotide comprises an mRNA encoding apolypeptide contemplated herein. In certain embodiments, the mRNAcomprises a cap, one or more nucleotides, and a poly(A) tail.

Terms that describe the orientation of polynucleotides include: 5′(normally the end of the polynucleotide having a free phosphate group)and 3′ (normally the end of the polynucleotide having a free hydroxyl(OH) group). Polynucleotide sequences can be annotated in the 5′ to 3′orientation or the 3′ to 5′ orientation. For DNA and mRNA, the 5′ to 3′strand is designated the “sense,” “plus,” or “coding” strand because itssequence is identical to the sequence of the premessenger (premRNA)[except for uracil (U) in RNA, instead of thymine (T) in DNA]. For DNAand mRNA, the complementary 3′ to 5′ strand which is the strandtranscribed by the RNA polymerase is designated as “template,”“antisense,” “minus,” or “non-coding” strand. As used herein, the term“reverse orientation” refers to a 5′ to 3′ sequence written in the 3′ to5′ orientation or a 3′ to 5′ sequence written in the 5′ to 3′orientation.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the complementary strand of the DNA sequence 5′ A G T C A T G3′ is 3′ T C A G T A C 5′. The latter sequence is often written as thereverse complement with the 5′ end on the left and the 3′ end on theright, 5′ C A T G A C T 3′. A sequence that is equal to its reversecomplement is said to be a palindromic sequence. Complementarity can be“partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there can be “complete” or“total” complementarity between the nucleic acids.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide, or fragment of variantthereof, as described herein. Some of these polynucleotides bear minimalhomology to the nucleotide sequence of any native gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated in particular embodiments, for examplepolynucleotides that are optimized for human and/or primate codonselection. In particular embodiments, the polynucleotides are codonoptimized for expression and/or stability. Further, alleles of the genescomprising the polynucleotide sequences provided herein may also beused. Alleles are endogenous genes that are altered as a result of oneor more mutations, such as deletions, additions and/or substitutions ofnucleotides.

The term “nucleic acid cassette” or “expression cassette” as used hereinrefers to genetic sequences within the vector which can express an RNA,and subsequently a polypeptide. In one embodiment, the nucleic acidcassette contains a gene(s)-of-interest, e.g., apolynucleotide(s)-of-interest. In another embodiment, the nucleic acidcassette contains one or more expression control sequences, e.g., apromoter, enhancer, poly(A) sequence, and a gene(s)-of-interest, e.g., apolynucleotide(s)-of-interest. Vectors may comprise 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 or more nucleic acid cassettes. The nucleic acid cassette ispositionally and sequentially oriented within the vector such that thenucleic acid in the cassette can be transcribed into RNA, and whennecessary, translated into a protein or a polypeptide, undergoappropriate post-translational modifications required for activity inthe transformed cell, and be translocated to the appropriate compartmentfor biological activity by targeting to appropriate intracellularcompartments or secretion into extracellular compartments. Preferably,the cassette has its 3′ and 5′ ends adapted for ready insertion into avector, e.g., it has restriction endonuclease sites at each end. In apreferred embodiment, the nucleic acid cassette contains the sequence ofa therapeutic gene used to treat, prevent, or ameliorate a geneticdisorder. The cassette can be removed and inserted into a plasmid orviral vector as a single unit.

Polynucleotides include polynucleotide(s)-of-interest. As used herein,the term “polynucleotide-of-interest” refers to a polynucleotideencoding a polypeptide or fusion polypeptide or a polynucleotide thatserves as a template for the transcription of an inhibitorypolynucleotide, as contemplated herein.

The polynucleotides contemplated herein, regardless of the length of thecoding sequence itself, may be combined with other DNA sequences, suchas promoters and/or enhancers, untranslated regions (UTRs), signalsequences, Kozak sequences, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, internal ribosomalentry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, andAtt sites), termination codons, transcriptional termination signals, andpolynucleotides encoding self-cleaving polypeptides, epitope tags, asdisclosed elsewhere herein or as known in the art, such that theiroverall length may vary considerably. It is therefore contemplated thata polynucleotide fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

Polynucleotides can be prepared, manipulated, expressed and/or deliveredusing any of a variety of well-established techniques known andavailable in the art. In order to express a desired polypeptide, anucleotide sequence encoding the polypeptide, can be inserted intoappropriate vector.

Illustrative examples of vectors include, but are not limited toplasmid, autonomously replicating sequences, and transposable elements,e.g., Sleeping Beauty, PiggyBac.

Additional Illustrative examples of vectors include, without limitation,plasmids, phagemids, cosmids, artificial chromosomes such as yeastartificial chromosome (YAC), bacterial artificial chromosome (BAC), orP1-derived artificial chromosome (PAC), bacteriophages such as lambdaphage or M13 phage, and animal viruses.

Illustrative examples of viruses useful as vectors include, withoutlimitation, retrovirus (including lentivirus), adenovirus,adeno-associated virus, herpesvirus (e.g., herpes simplex virus),poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40).

Illustrative examples of expression vectors include but are not limitedto pClneo vectors (Promega) for expression in mammalian cells;pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ(Invitrogen) for lentivirus-mediated gene transfer and expression inmammalian cells. In particular embodiments, coding sequences ofpolypeptides disclosed herein can be ligated into such expressionvectors for the expression of the polypeptides in mammalian cells.

In particular embodiments, the vector is an episomal vector or a vectorthat is maintained extrachromosomally. As used herein, the term“episomal” refers to a vector that is able to replicate withoutintegration into host's chromosomal DNA and without gradual loss from adividing host cell also meaning that said vector replicatesextrachromosomally or episomally.

“Expression control sequences,” “control elements,” or “regulatorysequences” present in an expression vector are those non-translatedregions of the vector including but not limited to the origin ofreplication, selection cassettes, promoters, enhancers, translationinitiation signals (Shine Dalgarno sequence or Kozak sequence) introns,a polyadenylation sequence, 5′ and 3′ untranslated regions, all of whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including ubiquitous promotersand inducible promoters may be used.

In particular embodiments, a polynucleotide comprises a vector,including but not limited to expression vectors and viral vectors. Avector may comprise one or more exogenous, endogenous, or heterologouscontrol sequences such as promoters and/or enhancers. An “endogenouscontrol sequence” is one which is naturally linked with a given gene inthe genome. An “exogenous control sequence” is one which is placed injuxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques) such that transcription of that gene isdirected by the linked enhancer/promoter. A “heterologous controlsequence” is an exogenous sequence that is from a different species thanthe cell being genetically manipulated. A “synthetic” control sequencemay comprise elements of one more endogenous and/or exogenous sequences,and/or sequences determined in vitro or in silico that provide optimalpromoter and/or enhancer activity for the particular therapy.

The term “promoter” as used herein refers to a recognition site of apolynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNApolymerase initiates and transcribes polynucleotides operably linked tothe promoter. In particular embodiments, promoters operative inmammalian cells comprise an AT-rich region located approximately 25 to30 bases upstream from the site where transcription is initiated and/oranother sequence found 70 to 80 bases upstream from the start oftranscription, a CNCAAT region where N may be any nucleotide.

The term “enhancer” refers to a segment of DNA which contains sequencescapable of providing enhanced transcription and in some instances canfunction independent of their orientation relative to another controlsequence. An enhancer can function cooperatively or additively withpromoters and/or other enhancer elements. The term “promoter/enhancer”refers to a segment of DNA which contains sequences capable of providingboth promoter and enhancer functions.

The term “operably linked”, refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. In one embodiment, the term refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, and/or enhancer) and a second polynucleotidesequence, e.g., a polynucleotide-of-interest, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

As used herein, the term “constitutive expression control sequence”refers to a promoter, enhancer, or promoter/enhancer that continually orcontinuously allows for transcription of an operably linked sequence. Aconstitutive expression control sequence may be a “ubiquitous” promoter,enhancer, or promoter/enhancer that allows expression in a wide varietyof cell and tissue types or a “cell specific,” “cell type specific,”“cell lineage specific,” or “tissue specific” promoter, enhancer, orpromoter/enhancer that allows expression in a restricted variety of celland tissue types, respectively.

Illustrative ubiquitous expression control sequences suitable for use inparticular embodiments include, but are not limited to, acytomegalovirus (CMV) immediate early promoter, a viral simian virus 40(SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV)LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus(HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters fromvaccinia virus, an elongation factor 1-alpha (EF1a) promoter, earlygrowth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL),Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translationinitiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5),heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein70 kDa (HSP70), β-kinesin (β-KIN), the human ROSA 26 locus (Irions etal., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C promoter(UBC), a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirusenhancer/chicken β-actin (CAG) promoter, a β-actin promoter and amyeloproliferative sarcoma virus enhancer, negative control regiondeleted, dl587rev primer-binding site substituted (MND) U3 promoter(Haas et al. Journal of Virology. 2003; 77(17): 9439-9450).

In one embodiment, a vector comprises an MNDU3 promoter.

In one embodiment, a vector comprises an EF1a promoter comprising thefirst intron of the human EF1a gene.

In one embodiment, a vector comprises an EF1a promoter that lacks thefirst intron of the human EF1a gene.

In a particular embodiment, it may be desirable to use a cell, celltype, cell lineage or tissue specific expression control sequence toachieve cell type specific, lineage specific, or tissue specificexpression of a desired polynucleotide sequence (e.g., to express aparticular nucleic acid encoding a polypeptide in only a subset of celltypes, cell lineages, or tissues or during specific stages ofdevelopment).

In a particular embodiment, it may be desirable to express apolynucleotide a T cell specific promoter.

As used herein, “conditional expression” may refer to any type ofconditional expression including, but not limited to, inducibleexpression; repressible expression; expression in cells or tissueshaving a particular physiological, biological, or disease state, etc.This definition is not intended to exclude cell type or tissue specificexpression. Certain embodiments provide conditional expression of apolynucleotide-of-interest, e.g., expression is controlled by subjectinga cell, tissue, organism, etc., to a treatment or condition that causesthe polynucleotide to be expressed or that causes an increase ordecrease in expression of the polynucleotide encoded by thepolynucleotide-of-interest.

Illustrative examples of inducible promoters/systems include, but arenot limited to, steroid-inducible promoters such as promoters for genesencoding glucocorticoid or estrogen receptors (inducible by treatmentwith the corresponding hormone), metallothionine promoter (inducible bytreatment with various heavy metals), MX-1 promoter (inducible byinterferon), the “GeneSwitch” mifepristone-regulatable system (Sirin etal., 2003, Gene, 323:67), the cumate inducible gene switch (WO2002/088346), tetracycline-dependent regulatory systems, etc. Induceragents include, but are not limited to glucocorticoids, estrogens,mifepristone (RU486), metals, interferons, small molecules, cumate,tetracycline, doxycycline, and variants thereof.

Conditional expression can also be achieved by using a site specific DNArecombinase. According to certain embodiments the vector comprises atleast one (typically two) site(s) for recombination mediated by a sitespecific recombinase. As used herein, the terms “recombinase” or “sitespecific recombinase” include excisive or integrative proteins, enzymes,co-factors or associated proteins that are involved in recombinationreactions involving one or more recombination sites (e.g., two, three,four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.),which may be wild-type proteins (see Landy, Current Opinion inBiotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusionproteins containing the recombination protein sequences or fragmentsthereof), fragments, and variants thereof. Illustrative examples ofrecombinases suitable for use in particular embodiments include, but arenot limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

The polynucleotides may comprise one or more recombination sites for anyof a wide variety of site specific recombinases. It is to be understoodthat the target site for a site specific recombinase is in addition toany site(s) required for integration of a vector, e.g., a retroviralvector or lentiviral vector. As used herein, the terms “recombinationsequence,” “recombination site,” or “site specific recombination site”refer to a particular nucleic acid sequence to which a recombinaserecognizes and binds.

For example, one recombination site for Cre recombinase is loxP which isa 34 base pair sequence comprising two 13 base pair inverted repeats(serving as the recombinase binding sites) flanking an 8 base pair coresequence (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology5:521-527 (1994)). Other exemplary loxP sites include but are notlimited to: lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171(Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al.,2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

Suitable recognition sites for the FLP recombinase include, but are notlimited to: FRT (McLeod, et al., 1996), F₁, F₂, F₃ (Schlake and Bode,1994), F₄, F₅ (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988),FRT(RE) (Senecoff et al., 1988).

Other examples of recognition sequences are the attB, attP, attL, andattR sequences, which are recognized by the recombinase enzyme λIntegrase, e.g., phi-c31. The φC31 SSR mediates recombination onlybetween the heterotypic sites attB (34 bp in length) and attP (39 bp inlength) (Groth et al., 2000). attB and attP, named for the attachmentsites for the phage integrase on the bacterial and phage genomes,respectively, both contain imperfect inverted repeats that are likelybound by φC31 homodimers (Groth et al., 2000). The product sites, attLand attR, are effectively inert to further φC31-mediated recombination(Belteki et al., 2003), making the reaction irreversible. For catalyzinginsertions, it has been found that attB-bearing DNA inserts into agenomic attP site more readily than an attP site into a genomic attBsite (Thyagaraj an et al., 2001; Belteki et al., 2003). Thus, typicalstrategies position by homologous recombination an attP-bearing “dockingsite” into a defined locus, which is then partnered with an attB-bearingincoming sequence for insertion.

As used herein, an “internal ribosome entry site” or “IRES” refers to anelement that promotes direct internal ribosome entry to the initiationcodon, such as ATG, of a cistron (a protein encoding region), therebyleading to the cap-independent translation of the gene. See, e.g.,Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson andKaminski. 1995. RNA 1(10):985-1000. Examples of IRES generally employedby those of skill in the art include those described in U.S. Pat. No.6,692,736. Further examples of “IRES” known in the art include but arenot limited to IRES obtainable from picornavirus (Jackson et al., 1990)and IRES obtainable from viral or cellular mRNA sources, such as forexample, immunoglobulin heavy-chain binding protein (BiP), the vascularendothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol.18(11):6178-6190), the fibroblast growth factor 2 (FGF-2), andinsulin-like growth factor (IGFII), the translational initiation factoreIF4G and yeast transcription factors TFIID and HAP4, theencephelomycarditis virus (EMCV) which is commercially available fromNovagen (Duke et al., 1992. J. Virol 66(3):1602-9) and the VEGF IRES(Huez et al., 1998. Mol Cell Biol 18(11):6178-90). IRES have also beenreported in viral genomes of Picornaviridae, Dicistroviridae andFlaviviridae species and in HCV, Friend murine leukemia virus (FrMLV)and Moloney murine leukemia virus (MoMLV).

In one embodiment, the IRES used in polynucleotides contemplated hereinis an EMCV IRES.

In particular embodiments, the polynucleotides comprise polynucleotidesthat have a consensus Kozak sequence and that encode a desiredpolypeptide. As used herein, the term “Kozak sequence” refers to a shortnucleotide sequence that greatly facilitates the initial binding of mRNAto the small subunit of the ribosome and increases translation. Theconsensus Kozak sequence is (GCC)RCCATGG (SEQ ID NO:43), where R is apurine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987.Nucleic Acids Res. 15(20):8125-48).

Elements directing the efficient termination and polyadenylation of theheterologous nucleic acid transcripts increases heterologous geneexpression. Transcription termination signals are generally founddownstream of the polyadenylation signal. In particular embodiments,vectors comprise a polyadenylation sequence 3′ of a polynucleotideencoding a polypeptide to be expressed. The term “polyA site” or “polyAsequence” as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript by RNApolymerase II. Polyadenylation sequences can promote mRNA stability byaddition of a polyA tail to the 3′ end of the coding sequence and thus,contribute to increased translational efficiency. Cleavage andpolyadenylation is directed by a poly(A) sequence in the RNA. The corepoly(A) sequence for mammalian pre-mRNAs has two recognition elementsflanking a cleavage-polyadenylation site. Typically, an almost invariantAAUAAA hexamer lies 20-50 nucleotides upstream of a more variableelement rich in U or GU residues. Cleavage of the nascent transcriptoccurs between these two elements and is coupled to the addition of upto 250 adenosines to the 5′ cleavage product. In particular embodiments,the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA,ATTAAA, AGTAAA). In particular embodiments, the poly(A) sequence is anSV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), arabbit β-globin polyA sequence (rβgpA), variants thereof, or anothersuitable heterologous or endogenous polyA sequence known in the art. Inparticular embodiments, the poly(A) sequence is synthetic.

In some embodiments, a polynucleotide or cell harboring thepolynucleotide utilizes a suicide gene, including an inducible suicidegene to reduce the risk of direct toxicity and/or uncontrolledproliferation. In specific embodiments, the suicide gene is notimmunogenic to the host harboring the polynucleotide or cell. A certainexample of a suicide gene that may be used is caspase-9 or caspase-8 orcytosine deaminase. Caspase-9 can be activated using a specific chemicalinducer of dimerization (CID).

In certain embodiments, polynucleotides comprise gene segments thatcause the immune effector cells, e.g., T cells, to be susceptible tonegative selection in vivo. By “negative selection” is meant that theinfused cell can be eliminated as a result of a change in the in vivocondition of the individual. The negative selectable phenotype mayresult from the insertion of a gene that confers sensitivity to anadministered agent, for example, a compound. Negative selectable genesare known in the art, and include, inter alia the following: the Herpessimplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al.,Cell 11:223, 1977) which confers ganciclovir sensitivity; the cellularhypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adeninephosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase,(Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some embodiments, genetically modified immune effector cells, such asT cells, comprise a polynucleotide further comprising a positive markerthat enables the selection of cells of the negative selectable phenotypein vitro. The positive selectable marker may be a gene which, upon beingintroduced into the host cell expresses a dominant phenotype permittingpositive selection of cells carrying the gene. Genes of this type areknown in the art, and include, inter alia, hygromycin-Bphosphotransferase gene (hph) which confers resistance to hygromycin B,the amino glycoside phosphotransferase gene (neo or aph) from Tn5 whichcodes for resistance to the antibiotic G418, the dihydrofolate reductase(DHFR) gene, the adenosine deaminase gene (ADA), and the multi-drugresistance (MDR) gene.

In one embodiment, the positive selectable marker and the negativeselectable element are linked such that loss of the negative selectableelement necessarily also is accompanied by loss of the positiveselectable marker. In a particular embodiment, the positive and negativeselectable markers are fused so that loss of one obligatorily leads toloss of the other. An example of a fused polynucleotide that yields asan expression product a polypeptide that confers both the desiredpositive and negative selection features described above is a hygromycinphosphotransferase thymidine kinase fusion gene (HyTK). Expression ofthis gene yields a polypeptide that confers hygromycin B resistance forpositive selection in vitro, and ganciclovir sensitivity for negativeselection in vivo. See also the publications of PCT US91/08442 andPCT/US94/05601, by S. D. Lupton, describing the use of bifunctionalselectable fusion genes derived from fusing a dominant positiveselectable markers with negative selectable markers.

Preferred positive selectable markers are derived from genes selectedfrom the group consisting of hph, nco, and gpt, and preferred negativeselectable markers are derived from genes selected from the groupconsisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt.Exemplary bifunctional selectable fusion genes contemplated inparticular embodiments include but are not limited to genes wherein thepositive selectable marker is derived from hph or neo, and the negativeselectable marker is derived from cytosine deaminase or a TK gene orselectable marker.

In particular embodiments, polynucleotides encoding one or morepolypeptides, or fusion polypeptides may be introduced into immuneeffector cells, e.g., T cells, by both non-viral and viral methods. Inparticular embodiments, delivery of one or more polynucleotides may beprovided by the same method or by different methods, and/or by the samevector or by different vectors.

The term “vector” is used herein to refer to a nucleic acid moleculecapable transferring or transporting another nucleic acid molecule. Thetransferred nucleic acid is generally linked to, e.g., inserted into,the vector nucleic acid molecule. A vector may include sequences thatdirect autonomous replication in a cell or may include sequencessufficient to allow integration into host cell DNA. In particularembodiments, non-viral vectors are used to deliver one or morepolynucleotides contemplated herein to a T cell.

Illustrative examples of non-viral vectors include, but are not limitedto plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids,and bacterial artificial chromosomes.

Illustrative methods of non-viral delivery of polynucleotidescontemplated in particular embodiments include, but are not limited to:electroporation, sonoporation, lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, nanoparticles, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions,DEAE-dextran-mediated transfer, gene gun, and heat-shock.

Illustrative examples of polynucleotide delivery systems suitable foruse in particular embodiments contemplated in particular embodimentsinclude, but are not limited to those provided by Amaxa Biosystems,Maxcyte, Inc., BTX Molecular Delivery Systems, and CopernicusTherapeutics Inc. Lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides have been described in the literature. See e.g., Liu etal. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal ofDrug Delivery. 2011:1-12. Antibody-targeted, bacterially derived,non-living nanocell-based delivery is also contemplated in particularembodiments.

Viral vectors comprising polynucleotides contemplated in particularembodiments can be delivered in vivo by administration to an individualpatient, typically by systemic administration (e.g., intravenous,intraperitoneal, intramuscular, subdermal, or intracranial infusion) ortopical application, as described below. Alternatively, vectors can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., mobilized peripheral blood, lymphocytes, bone marrowaspirates, tissue biopsy, etc.) or universal donor hematopoietic stemcells, followed by reimplantation of the cells into a patient.

In one embodiment, viral vectors comprising polynucleotides contemplatedherein are administered directly to an organism for transduction ofcells in vivo. Alternatively, naked DNA can be administered.Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells including, butnot limited to, injection, infusion, topical application andelectroporation. Suitable methods of administering such nucleic acidsare available and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Illustrative examples of viral vector systems suitable for use inparticular embodiments contemplated in particular embodiments includebut are not limited to adeno-associated virus (AAV), retrovirus, herpessimplex virus, adenovirus, and vaccinia virus vectors.

In various embodiments, one or more polynucleotides are introduced intoan immune effector cell, e.g., T cell, by transducing the cell with arecombinant adeno-associated virus (rAAV), comprising the one or morepolynucleotides.

AAV is a small (˜26 nm) replication-defective, primarily episomal,non-enveloped virus. AAV can infect both dividing and non-dividing cellsand may incorporate its genome into that of the host cell. RecombinantAAV (rAAV) are typically composed of, at a minimum, a transgene and itsregulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs). The ITR sequences are about 145 bp in length. In particularembodiments, the rAAV comprises ITRs and capsid sequences isolated fromAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

In some embodiments, a chimeric rAAV is used the ITR sequences areisolated from one AAV serotype and the capsid sequences are isolatedfrom a different AAV serotype. For example, a rAAV with ITR sequencesderived from AAV2 and capsid sequences derived from AAV6 is referred toas AAV2/AAV6. In particular embodiments, the rAAV vector may compriseITRs from AAV2, and capsid proteins from any one of AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment,the rAAV comprises ITR sequences derived from AAV2 and capsid sequencesderived from AAV6. In a preferred embodiment, the rAAV comprises ITRsequences derived from AAV2 and capsid sequences derived from AAV2.

In some embodiments, engineering and selection methods can be applied toAAV capsids to make them more likely to transduce cells of interest.

Construction of rAAV vectors, production, and purification thereof havebeen disclosed, e.g., in U.S. Pat. Nos. 9,169,494; 9,169,492; 9,012,224;8,889,641; 8,809,058; and 8,784,799, each of which is incorporated byreference herein, in its entirety.

In various embodiments, one or more polynucleotides are introduced intoan immune effector cell, e.g., T cell, by transducing the cell with aretrovirus, e.g., lentivirus, comprising the one or morepolynucleotides.

As used herein, the term “retrovirus” refers to an RNA virus thatreverse transcribes its genomic RNA into a linear double-stranded DNAcopy and subsequently covalently integrates its genomic DNA into a hostgenome. Illustrative retroviruses suitable for use in particularembodiments, include, but are not limited to: Moloney murine leukemiavirus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon apeleukemia virus (GaLV), feline leukemia virus (FLV), Spumavirus, Friendmurine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous SarcomaVirus (RSV)) and lentivirus.

As used herein, the term “lentivirus” refers to a group (or genus) ofcomplex retroviruses. Illustrative lentiviruses include but are notlimited to: HIV (human immunodeficiency virus; including HIV type 1, andHIV type 2); visna-maedi virus (VMV) virus; the caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (FIV); bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV). In one embodiment,HIV based vector backbones (i.e., HIV cis-acting sequence elements) arepreferred.

In various embodiments, a lentiviral vector contemplated hereincomprises one or more LTRs, and one or more, or all, of the followingaccessory elements: a cPPT/FLAP, a Psi (T) packaging signal, an exportelement, poly (A) sequences, and may optionally comprise a WPRE or HPRE,an insulator element, a selectable marker, and a cell suicide gene, asdiscussed elsewhere herein.

In particular embodiments, lentiviral vectors contemplated herein may beintegrative or non-integrating or integration defective lentivirus. Asused herein, the term “integration defective lentivirus” or “IDLV”refers to a lentivirus having an integrase that lacks the capacity tointegrate the viral genome into the genome of the host cells.Integration-incompetent viral vectors have been described in patentapplication WO 2006/010834, which is herein incorporated by reference inits entirety.

Illustrative mutations in the HIV-1 pol gene suitable to reduceintegrase activity include, but are not limited to: H12N, H12C, H16C,H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A,E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E,K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A,K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A,Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A,R262A, R263A and K264H.

The term “long terminal repeat (LTR)” refers to domains of base pairslocated at the ends of retroviral DNAs which, in their natural sequencecontext, are direct repeats and contain U3, R and U5 regions.

As used herein, the term “FLAP element” or “cPPT/FLAP” refers to anucleic acid whose sequence includes the central polypurine tract andcentral termination sequences (cPPT and CTS) of a retrovirus, e.g.,HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No.6,682,907 and in Zennou, et al., 2000, Cell, 101:173.

As used herein, the term “packaging signal” or “packaging sequence”refers to psi NI sequences located within the retroviral genome whichare required for insertion of the viral RNA into the viral capsid orparticle, see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4;pp. 2101-2109.

The term “export element” refers to a cis-acting post-transcriptionalregulatory element which regulates the transport of an RNA transcriptfrom the nucleus to the cytoplasm of a cell. Examples of RNA exportelements include, but are not limited to, the human immunodeficiencyvirus (HIV) rev response element (RRE) (see e.g., Cullen et al., 1991.J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and thehepatitis B virus post-transcriptional regulatory element (HPRE).

In particular embodiments, expression of heterologous sequences in viralvectors is increased by incorporating posttranscriptional regulatoryelements, efficient polyadenylation sites, and optionally, transcriptiontermination signals into the vectors. A variety of posttranscriptionalregulatory elements can increase expression of a heterologous nucleicacid at the protein, e.g., woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886);the posttranscriptional regulatory element present in hepatitis B virus(HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu etal., 1995, Genes Dev., 9:1766).

Lentiviral vectors preferably contain several safety enhancements as aresult of modifying the LTRs. “Self-inactivating” (SIN) vectors refersto replication-defective vectors, e.g., retroviral or lentiviralvectors, in which the right (3′) LTR enhancer-promoter region, known asthe U3 region, has been modified (e.g., by deletion or substitution) toprevent viral transcription beyond the first round of viral replication.Self-inactivation is preferably achieved through in the introduction ofa deletion in the U3 region of the 3′ LTR of the vector DNA, i.e., theDNA used to produce the vector RNA. Thus, during reverse transcription,this deletion is transferred to the 5′ LTR of the proviral DNA. Inparticular embodiments, it is desirable to eliminate enough of the U3sequence to greatly diminish or abolish altogether the transcriptionalactivity of the LTR, thereby greatly diminishing or abolishing theproduction of full-length vector RNA in transduced cells. In the case ofHIV based lentivectors, it has been discovered that such vectorstolerate significant U3 deletions, including the removal of the LTR TATAbox (e.g., deletions from −418 to −18), without significant reductionsin vector titers.

An additional safety enhancement is provided by replacing the U3 regionof the 5′ LTR with a heterologous promoter to drive transcription of theviral genome during production of viral particles. Examples ofheterologous promoters which can be used include, for example, viralsimian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV)(e.g., immediate early), Moloney murine leukemia virus (MoMLV), Roussarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase)promoters.

The terms “pseudotype” or “pseudotyping” as used herein, refer to avirus whose viral envelope proteins have been substituted with those ofanother virus possessing preferable characteristics. For example, HIVcan be pseudotyped with vesicular stomatitis virus G-protein (VSV-G)envelope proteins, which allows HIV to infect a wider range of cellsbecause HIV envelope proteins (encoded by the env gene) normally targetthe virus to CD4⁺ presenting cells.

In certain embodiments, lentiviral vectors are produced according toknown methods. See e.g., Kutner et al., BMC Biotechnol. 2009; 9:10. doi:10.1186/1472-6750-9-10; Kutner et al. Nat. Protoc. 2009; 4(4):495-505.doi: 10.1038/nprot.2009.22.

According to certain specific embodiments contemplated herein, most orall of the viral vector backbone sequences are derived from alentivirus, e.g., HIV-1. However, it is to be understood that manydifferent sources of retroviral and/or lentiviral sequences can be usedor combined and numerous substitutions and alterations in certain of thelentiviral sequences may be accommodated without impairing the abilityof a transfer vector to perform the functions described herein.Moreover, a variety of lentiviral vectors are known in the art, seeNaldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dullet al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136, many of which maybe adapted to produce a viral vector or transfer plasmid contemplatedherein.

In various embodiments, one or more polynucleotides are introduced intoan immune effector cell, by transducing the cell with an adenoviruscomprising the one or more polynucleotides.

Adenoviral based vectors are capable of very high transductionefficiency in many cell types and do not require cell division. Withsuch vectors, high titer and high levels of expression have beenobtained. This vector can be produced in large quantities in arelatively simple system. Most adenovirus vectors are engineered suchthat a transgene replaces the Ad E1a, E1b, and/or E3 genes; subsequentlythe replication defective vector is propagated in human 293 cells thatsupply deleted gene function in trans. Ad vectors can transduce multipletypes of tissues in vivo, including non-dividing, differentiated cellssuch as those found in liver, kidney and muscle. Conventional Ad vectorshave a large carrying capacity.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, may utilize a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones & Shenk, 1978), the current adenovirus vectors, with the help of293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham & Prevec, 1991). Adenovirus vectors have been used in eukaryoticgene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) andvaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992).Studies in administering recombinant adenovirus to different tissuesinclude trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al.,1992), muscle injection (Ragot et al., 1993), peripheral intravenousinjections (Herz & Gerard, 1993) and stereotactic inoculation into thebrain (Le Gal La Salle et al., 1993). An example of the use of an Advector in a clinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)).

In various embodiments, one or more polynucleotides are introduced intoan immune effector cell by transducing the cell with a herpes simplexvirus, e.g., HSV-1, HSV-2, comprising the one or more polynucleotides.

The mature HSV virion consists of an enveloped icosahedral capsid with aviral genome consisting of a linear double-stranded DNA molecule that is152 kb. In one embodiment, the HSV based viral vector is deficient inone or more essential or non-essential HSV genes. In one embodiment, theHSV based viral vector is replication deficient. Most replicationdeficient HSV vectors contain a deletion to remove one or moreintermediate-early, early, or late HSV genes to prevent replication. Forexample, the HSV vector may be deficient in an immediate early geneselected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and acombination thereof. Advantages of the HSV vector are its ability toenter a latent stage that can result in long-term DNA expression and itslarge viral DNA genome that can accommodate exogenous DNA inserts of upto 25 kb. HSV-based vectors are described in, for example, U.S. Pat.Nos. 5,837,532, 5,846,782, and 5,804,413, and International PatentApplications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583,each of which are incorporated by reference herein in its entirety.

G. Genetically Modified Cells

In various embodiments, cells are modified to express DARIC immunereceptors, engineered TCRs, CARs, zetakines, and/or fusion proteinscontemplated herein, for use in the treatment of cancer. Cells may benon-genetically modified to express the polypeptides contemplatedherein, or in particular preferred embodiments, cells may be geneticallymodified to express the polypeptides contemplated herein. As usedherein, the term “genetically engineered” or “genetically modified”refers to the addition of extra genetic material in the form of DNA orRNA into the total genetic material in a cell. The terms, “geneticallymodified cells,” “modified cells,” and “redirected cells,” are usedinterchangeably in particular embodiments.

In particular embodiments, the DARIC immune receptor polypeptidescontemplated herein are introduced and expressed in immune effectorcells to improve the resistance of the cells to the immunosuppressivesignals in the TME. In particular embodiments, DARIC immune receptorpolypeptides are introduced and expressed in immune effector cells thathave been redirected to a target cell by virtue of co-expressing anengineered antigen receptor in the cell.

An “immune effector cell,” is any cell of the immune system that has oneor more effector functions (e.g., cytotoxic cell killing activity,secretion of cytokines, induction of ADCC and/or CDC). The illustrativeimmune effector cells contemplated herein are T lymphocytes, inparticular cytotoxic T cells (CTLs; CD8+ T cells), TILs, and helper Tcells (HTLs; CD4+ T cells. In one embodiment, immune effector cellsinclude natural killer (NK) cells. In one embodiment, immune effectorcells include natural killer T (NKT) cells. Immune effector cells can beautologous/autogeneic (“self”) or non-autologous (“non-self,” e.g.,allogeneic, syngeneic or xenogeneic).

“Autologous,” as used herein, refers to cells from the same subject.“Allogeneic,” as used herein, refers to cells of the same species thatdiffer genetically to the cell in comparison. “Syngeneic,” as usedherein, refers to cells of a different subject that are geneticallyidentical to the cell in comparison. “Xenogeneic,” as used herein,refers to cells of a different species to the cell in comparison. Inpreferred embodiments, the cells are autologous.

Illustrative immune effector cells suitable for introducing the DARICimmune receptor polypeptides contemplated herein include T lymphocytes.The terms “T cell” or “T lymphocyte” are art-recognized and are intendedto include thymocytes, immature T lymphocytes, mature T lymphocytes,resting T lymphocytes, or activated T lymphocytes. A T cell can be a Thelper (Th) cell, for example a T helper 1 (Th1) or a T helper 2 (Th2)cell. The T cell can be a helper T cell (HTL; CD4⁺ T cell) CD4⁺ T cell,a cytotoxic T cell (CTL; CD8⁺ T cell), CD4⁺CD8⁺ T cell, CD4⁻CD8⁻ T cell,or any other subset of T cells. Other illustrative populations of Tcells suitable for use in particular embodiments include naïve T cellsand memory T cells.

As would be understood by the skilled person, other cells may also beused as immune effector cells with DARIC immune receptor polypeptidescontemplated herein. In particular, immune effector cells also includeNK cells, NKT cells, neutrophils, and macrophages. Immune effector cellsalso include progenitors of effector cells wherein such progenitor cellscan be induced to differentiate into immune effector cells in vivo or invitro. Thus, in particular embodiments, immune effector cell includesprogenitors of immune effectors cells such as hematopoietic stem cells(HSCs) contained within the CD34⁺ population of cells derived from cordblood, bone marrow or mobilized peripheral blood which uponadministration in a subject differentiate into mature immune effectorcells, or which can be induced in vitro to differentiate into matureimmune effector cells.

As used herein, immune effector cells genetically engineered to containa specific chimeric receptor may be referred to as, “antigen specificredirected immune effector cells.”

The term, “CD34⁺ cell,” as used herein refers to a cell expressing theCD34 protein on its cell surface. “CD34,” as used herein refers to acell surface glycoprotein (e.g., sialomucin protein) that often acts asa cell-cell adhesion factor and is involved in T cell entrance intolymph nodes. The CD34⁺ cell population contains hematopoietic stem cells(HSC), which upon administration to a patient differentiate andcontribute to all hematopoietic lineages, including T cells, NK cells,NKT cells, neutrophils and cells of the monocyte/macrophage lineage.

Methods for making the immune effector cells which express one or moreDARIC immune receptor polypeptides contemplated herein are provided inparticular embodiments. In one embodiment, the method comprisestransfecting or transducing immune effector cells isolated from anindividual such that the immune effector cells with one or more nucleicacids and/or vectors or combination thereof comprising one or more DARICimmune receptor polypeptides as contemplated herein. In one embodiment,the method comprises transfecting or transducing immune effector cellsisolated from an individual such that the immune effector cells expressone or more DARIC immune receptor polypeptides and engineered antigenreceptors contemplated herein. In certain embodiments, the immuneeffector cells are isolated from an individual and genetically modifiedwithout further manipulation in vitro. Such cells can then be directlyre-administered into the individual. In further embodiments, the immuneeffector cells are first activated and stimulated to proliferate invitro prior to being genetically modified. In this regard, the immuneeffector cells may be cultured before and/or after being geneticallymodified.

In particular embodiments, prior to in vitro manipulation or geneticmodification of the immune effector cells described herein, the sourceof cells is obtained from a subject. In particular embodiments, themodified immune effector cells comprise T cells.

T cells can be obtained from a number of sources including, but notlimited to, peripheral blood mononuclear cells, bone marrow, lymph nodestissue, cord blood, thymus issue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. In certainembodiments, T cells can be obtained from a unit of blood collected froma subject using any number of techniques known to the skilled person,such as sedimentation, e.g., FICOLL™ separation.

In other embodiments, an isolated or purified population of T cells isused. In some embodiments, after isolation of PBMC, both cytotoxic andhelper T lymphocytes can be sorted into naïve, memory, and effector Tcell subpopulations either before or after activation, expansion, and/orgenetic modification.

In one embodiment, an isolated or purified population of T cellsexpresses one or more of the markers including, but not limited to aCD3⁺, CD4⁺, CD8⁺, or a combination thereof.

In certain embodiments, the T cells are isolated from an individual andfirst activated and stimulated to proliferate in vitro prior to beingmodified to express one or more DARIC immune receptor polypeptides.

In order to achieve sufficient therapeutic doses of T cell compositions,T cells are often subjected to one or more rounds of stimulation,activation and/or expansion. T cells can be activated and expandedgenerally using methods as described, for example, in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which isincorporated herein by reference in its entirety. In particularembodiments, T cells are activated and expanded for about 6 hours, about12 hours, about 18 hours or about 24 hours prior to introduction ofvectors or polynucleotides encoding one or more DARIC immune receptorpolypeptides. Optionally in combination with an engineered antigenreceptor contemplated herein.

In one embodiment, T cells are activated at the same time that they aremodified.

In various embodiments, a method of generating an immune effector cellcomprises activating a population of cells comprising T cells andexpanding the population of T cells. T cell activation can beaccomplished by providing a primary stimulation signal through the Tcell TCR/CD3 complex and by providing a secondary co-stimulation signalthrough an accessory molecule, e.g., CD28.

The TCR/CD3 complex may be stimulated by contacting the T cell with asuitable CD3 binding agent, e.g., a CD3 ligand or an anti-CD3 monoclonalantibody. Illustrative examples of CD3 antibodies include, but are notlimited to, OKT3, G19-4, BC3, and 64.1.

In addition to the primary stimulation signal provided through theTCR/CD3 complex, induction of T cell responses requires a second,co-stimulatory signal. In particular embodiments, a CD28 binding agentcan be used to provide a co-stimulatory signal. Illustrative examples ofCD28 binding agents include but are not limited to: natural CD 28ligands, e.g., a natural ligand for CD28 (e.g., a member of the B7family of proteins, such as B7-1(CD80) and B7-2 (CD86); and anti-CD28monoclonal antibody or fragment thereof capable of crosslinking the CD28molecule, e.g., monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8,248.23.2, and EX5.3D10.

In one embodiment, the molecule providing the primary stimulationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex and the co-stimulatory molecule are coupled to the samesurface.

In certain embodiments, binding agents that provide stimulatory andco-stimulatory signals are localized on the surface of a cell. This canbe accomplished by transfecting or transducing a cell with a nucleicacid encoding the binding agent in a form suitable for its expression onthe cell surface or alternatively by coupling a binding agent to thecell surface.

In another embodiment, the molecule providing the primary stimulationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex and the co-stimulatory molecule are displayed on antigenpresenting cells.

In one embodiment, the molecule providing the primary stimulationsignal, for example a molecule which provides stimulation through theTCR/CD3 complex and the co-stimulatory molecule are provided on separatesurfaces.

In a certain embodiment, one of the binding agents that providesstimulatory and co-stimulatory signals is soluble (provided in solution)and the other agent(s) is provided on one or more surfaces.

In a particular embodiment, the binding agents that provide stimulatoryand co-stimulatory signals are both provided in a soluble form (providedin solution).

In various embodiments, the methods for making T cells contemplatedherein comprise activating T cells with anti-CD3 and anti-CD28antibodies.

In one embodiment, expanding T cells activated by the methodscontemplated herein further comprises culturing a population of cellscomprising T cells for several hours (about 3 hours) to about 7 days toabout 28 days or any hourly integer value in between. In anotherembodiment, the T cell composition may be cultured for 14 days. In aparticular embodiment, T cells are cultured for about 21 days. Inanother embodiment, the T cell compositions are cultured for about 2-3days. Several cycles of stimulation/activation/expansion may also bedesired such that culture time of T cells can be 60 days or more.

In particular embodiments, conditions appropriate for T cell cultureinclude an appropriate media (e.g., Minimal Essential Media or RPMIMedia 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary forproliferation and viability including, but not limited to serum (e.g.,fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ,IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or anyother additives suitable for the growth of cells known to the skilledartisan.

Further illustrative examples of cell culture media include, but are notlimited to RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15,and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.

Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

In particular embodiments, PBMCs or isolated T cells are contacted witha stimulatory agent and co-stimulatory agent, such as anti-CD3 andanti-CD28 antibodies, generally attached to a bead or other surface, ina culture medium with appropriate cytokines, such as IL-2, IL-7, and/orIL-15.

In other embodiments, artificial APC (aAPC) made by engineering K562,U937, 721.221, T2, and C1R cells to direct the stable expression andsecretion, of a variety of co-stimulatory molecules and cytokines. In aparticular embodiment K32 or U32 aAPCs are used to direct the display ofone or more antibody-based stimulatory molecules on the AAPC cellsurface. Populations of T cells can be expanded by aAPCs expressing avariety of co-stimulatory molecules including, but not limited to,CD137L (4-1BBL), CD134L (OX40L), and/or CD80 or CD86. Finally, the aAPCsprovide an efficient platform to expand genetically modified T cells andto maintain CD28 expression on CD8α T cells. aAPCs provided in WO03/057171 and US2003/0147869 are hereby incorporated by reference intheir entirety.

In a particular embodiment, polynucleotide encoding one or more DARICimmune receptor polypeptides and an engineered antigen receptor areintroduced into the population of T cells. In a particular embodiment,polynucleotide encoding one or more DARIC immune receptor polypeptidesis introduced into a population of T cells that express an engineeredantigen receptor. The polynucleotides may be introduced into the T cellsby microinjection, transfection, lipofection, heat-shock,electroporation, transduction, gene gun, microinjection,DEAE-dextran-mediated transfer, and the like.

In a preferred embodiment, polynucleotides are introduced into a T cellby viral transduction.

Illustrative examples of viral vector systems suitable for introducing apolynucleotide into an immune effector cell or CD34⁺ cell include butare not limited to adeno-associated virus (AAV), retrovirus, herpessimplex virus, adenovirus, vaccinia virus vectors for gene transfer.

In one embodiment, polynucleotides are introduced into a T cell by AAVtransduction.

In one embodiment, polynucleotides are introduced into a T cell byretroviral transduction.

In one embodiment, polynucleotides are introduced into a T cell bylentiviral transduction.

In one embodiment, polynucleotides are introduced into a T cell byadenovirus transduction.

In one embodiment, polynucleotides are introduced into a T cell byherpes simplex virus transduction.

In one embodiment, polynucleotides are introduced into a T cell byvaccinia virus transduction.

H. Compositions and Formulations

The compositions contemplated herein may comprise one or morepolypeptides, polynucleotides, vectors comprising same, geneticallymodified immune effector cells, bridging factors, etc. Compositionsinclude but are not limited to pharmaceutical compositions. A“pharmaceutical composition” refers to a composition formulated inpharmaceutically-acceptable or physiologically-acceptable solutions foradministration to a cell or an animal, either alone, or in combinationwith one or more other modalities of therapy. It will also be understoodthat, if desired, the compositions may be administered in combinationwith other agents as well, such as, e.g., cytokines, growth factors,hormones, small molecules, chemotherapeutics, pro-drugs, drugs,antibodies, or other various pharmaceutically-active agents. There isvirtually no limit to other components that may also be included in thecompositions, provided that the additional agents do not adverselyaffect the ability of the composition to deliver the intended therapy.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The term “pharmaceutically acceptable carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the bridging factors,polypeptides, polynucleotides, vectors comprising same, or geneticallymodified immune effector cells are administered. Illustrative examplesof pharmaceutical carriers can be sterile liquids, such as cell culturemedia, water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients inparticular embodiments, include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

In one embodiment, a composition comprising a pharmaceuticallyacceptable carrier is suitable for administration to a subject. Inparticular embodiments, a composition comprising a carrier is suitablefor parenteral administration, e.g., intravascular (intravenous orintraarterial), intraperitoneal or intramuscular administration. Inparticular embodiments, a composition comprising a pharmaceuticallyacceptable carrier is suitable for intraventricular, intraspinal, orintrathecal administration. Pharmaceutically acceptable carriers includesterile aqueous solutions, cell culture media, or dispersions. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the bridging factors, polypeptides, polynucleotides,vectors comprising same, or genetically modified immune effector cells,use thereof in the pharmaceutical compositions is contemplated.

In particular embodiments, compositions contemplated herein comprisegenetically modified T cells and a pharmaceutically acceptable carrier.A composition comprising a cell-based composition contemplated hereincan be administered separately by enteral or parenteral administrationmethods or in combination with other suitable compounds to effect thedesired treatment goals.

In particular embodiments, compositions contemplated herein comprise abridging factor and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier must be of sufficiently highpurity and of sufficiently low toxicity to render it suitable foradministration to the human subject being treated. It further shouldmaintain or increase the stability of the composition. Thepharmaceutically acceptable carrier can be liquid or solid and isselected, with the planned manner of administration in mind, to providefor the desired bulk, consistency, etc., when combined with othercomponents of the composition. For example, the pharmaceuticallyacceptable carrier can be, without limitation, a binding agent (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose, etc.), a filler (e.g., lactose and other sugars,microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant(e.g., magnesium stearate, talc, silica, colloidal silicon dioxide,stearic acid, metallic stearates, hydrogenated vegetable oils, cornstarch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), adisintegrant (e.g., starch, sodium starch glycolate, etc.), or a wettingagent (e.g., sodium lauryl sulfate, etc.). Other suitablepharmaceutically acceptable carriers for the compositions contemplatedherein include, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs,silicic acids, viscous paraffins, hydroxymethylcelluloses,polyvinylpyrrolidones and the like.

Such carrier solutions also can contain buffers, diluents and othersuitable additives. The term “buffer” as used herein refers to asolution or liquid whose chemical makeup neutralizes acids or baseswithout a significant change in pH. Examples of buffers contemplatedherein include, but are not limited to, Dulbecco's phosphate bufferedsaline (PBS), Ringer's solution, 5% dextrose in water (D5W),normal/physiologic saline (0.9% NaCl).

The pharmaceutically acceptable carriers may be present in amountssufficient to maintain a pH of the composition of about 7.Alternatively, the composition has a pH in a range from about 6.8 toabout 7.4, e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, and 7.4. In still anotherembodiment, the composition has a pH of about 7.4.

Compositions contemplated herein may comprise a nontoxicpharmaceutically acceptable medium. The compositions may be asuspension. The term “suspension” as used herein refers to non-adherentconditions in which cells are not attached to a solid support. Forexample, cells maintained as a suspension may be stirred or agitated andare not adhered to a support, such as a culture dish.

In particular embodiments, compositions contemplated herein areformulated in a suspension, where the modified T cells are dispersedwithin an acceptable liquid medium or solution, e.g., saline orserum-free medium, in an intravenous (IV) bag or the like. Acceptablediluents include, but are not limited to water, PlasmaLyte, Ringer'ssolution, isotonic sodium chloride (saline) solution, serum-free cellculture medium, and medium suitable for cryogenic storage, e.g.,Cryostor® medium.

In certain embodiments, a pharmaceutically acceptable carrier issubstantially free of natural proteins of human or animal origin, andsuitable for storing a composition comprising a population of modified Tcells. The therapeutic composition is intended to be administered into ahuman patient, and thus is substantially free of cell culture componentssuch as bovine serum albumin, horse serum, and fetal bovine serum.

In some embodiments, compositions are formulated in a pharmaceuticallyacceptable cell culture medium. Such compositions are suitable foradministration to human subjects. In particular embodiments, thepharmaceutically acceptable cell culture medium is a serum free medium.

Serum-free medium has several advantages over serum containing medium,including a simplified and better defined composition, a reduced degreeof contaminants, elimination of a potential source of infectious agents,and lower cost. In various embodiments, the serum-free medium isanimal-free, and may optionally be protein-free. Optionally, the mediummay contain biopharmaceutically acceptable recombinant proteins.“Animal-free” medium refers to medium wherein the components are derivedfrom non-animal sources. Recombinant proteins replace native animalproteins in animal-free medium and the nutrients are obtained fromsynthetic, plant or microbial sources. “Protein-free” medium, incontrast, is defined as substantially free of protein.

Illustrative examples of serum-free media used in particularcompositions includes but is not limited to QBSF-60 (Quality Biological,Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

In a preferred embodiment, the compositions comprising modified T cellsare formulated in PlasmaLyte.

In various embodiments, compositions comprising modified T cells areformulated in a cryopreservation medium. For example, cryopreservationmedia with cryopreservation agents may be used to maintain a high cellviability outcome post-thaw. Illustrative examples of cryopreservationmedia used in particular compositions includes, but is not limited to,CryoStor CS10, CryoStor CSS, and CryoStor CS2.

In one embodiment, the compositions are formulated in a solutioncomprising 50:50 PlasmaLyte A to CryoStor CS10.

In particular embodiments, the composition is substantially free ofmycoplasma, endotoxin, and microbial contamination. By “substantiallyfree” with respect to endotoxin is meant that there is less endotoxinper dose of cells than is allowed by the FDA for a biologic, which is atotal endotoxin of 5 EU/kg body weight per day, which for an average 70kg person is 350 EU per total dose of cells. In particular embodiments,compositions contemplated herein contain about 0.5 EU/mL to about 5.0EU/mL, or about 0.5 EU/mL, 1.0 EU/mL, 1.5 EU/mL, 2.0 EU/mL, 2.5 EU/mL,3.0 EU/mL, 3.5 EU/mL, 4.0 EU/mL, 4.5 EU/mL, or 5.0 EU/mL.

In particular embodiments, formulation of pharmaceutically-acceptablecarrier solutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., enteral and parenteral, e.g., intravascular,intravenous, intrarterial, intraosseously, intraventricular,intracerebral, intracranial, intraspinal, intrathecal, andintramedullary administration and formulation. It would be understood bythe skilled artisan that particular embodiments contemplated herein maycomprise other formulations, such as those that are well known in thepharmaceutical art, and are described, for example, in Remington: TheScience and Practice of Pharmacy, volume I and volume II. 22^(nd)Edition. Edited by Loyd V. Allen Jr. Philadelphia, Pa.: PharmaceuticalPress; 2012, which is incorporated by reference herein, in its entirety.

In particular embodiments, compositions comprise an amount of immuneeffector cells, including CAR T cells, that express one or more DARICimmune receptor polypeptides contemplated herein. As used herein, theterm “amount” refers to “an amount effective” or “an effective amount”of cells comprising one or more DARIC immune receptor polypeptidescontemplated herein, etc., to achieve a beneficial or desiredprophylactic or therapeutic result, including clinical results.

A “prophylactically effective amount” refers to an amount of cellscomprising one or more DARIC immune receptor polypeptides contemplatedherein, etc., effective to achieve the desired prophylactic result.Typically, but not necessarily, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount is less than the therapeuticallyeffective amount.

A “therapeutically effective amount” refers to an amount of cellscomprising one or more DARIC immune receptor polypeptides contemplatedherein that is effective to “treat” a subject (e.g., a patient). When atherapeutic amount is indicated, the precise amount of the compositionsto be administered can be determined by a physician with considerationof individual differences in age, weight, tumor size, extent ofinfection or metastasis, and condition of the patient (subject). It cangenerally be stated that a pharmaceutical composition comprising theimmune effector cells described herein may be administered at a dosageof 10² to 10¹⁰ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. The number ofcells will depend upon the ultimate use for which the composition isintended as will the type of cells included therein. For uses providedherein, the cells are generally in a volume of a liter or less, can be500 mLs or less, even 250 mLs or 100 mLs or less. Hence the density ofthe desired cells is typically greater than 10⁶ cells/ml and generallyis greater than 10⁷ cells/ml, generally 10⁸ cells/ml or greater. Theclinically relevant number of immune cells can be apportioned intomultiple infusions that cumulatively equal or exceed 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, or 10¹² cells. In some embodiments, particularly sinceall the infused cells will be redirected to a particular target antigen,lower numbers of cells, in the range of 10⁶/kilogram (10⁶-10¹¹ perpatient) may be administered. If desired, the treatment may also includeadministration of mitogens (e.g., PHA) or lymphokines, cytokines, and/orchemokines (e.g., IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta,GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein toenhance induction of the immune response.

Generally, compositions comprising the cells activated and expanded asdescribed herein may be utilized in the treatment and prevention ofdiseases that arise in individuals who are immunocompromised. Inparticular, compositions contemplated herein are used in the treatmentof cancer. In particular embodiments, the immune effector cells may beadministered either alone, or as a pharmaceutical compositions incombination with carriers, diluents, excipients, and/or with othercomponents such as IL-2 or other cytokines or cell populations.

In particular embodiments, pharmaceutical compositions comprise anamount of genetically modified T cells, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients.

In a particular embodiment, compositions comprise an effective amount ofimmune effector cells comprising one or more DARIC immune receptorpolypeptides contemplated herein, alone or in combination with one ormore therapeutic agents, such as radiation therapy, chemotherapy,transplantation, immunotherapy, hormone therapy, photodynamic therapy,etc. The compositions may also be administered in combination withantibiotics. Such therapeutic agents may be accepted in the art as astandard treatment for a particular disease state as described herein,such as a particular cancer. Exemplary therapeutic agents contemplatedinclude cytokines, growth factors, steroids, NSAIDs, DMARDs,anti-inflammatories, chemotherapeutics, radiotherapeutics, therapeuticantibodies, or other active and ancillary agents.

In certain embodiments, compositions comprising immune effector cellscomprising one or more DARIC immune receptor polypeptides contemplatedherein may be administered in conjunction with any number ofchemotherapeutic agents. Illustrative examples of chemotherapeuticagents include alkylating agents such as thiotepa and cyclophosphamide(CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoic acid derivatives such asTargretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on cancers such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

A variety of other therapeutic agents may be used in conjunction withthe compositions described herein. In one embodiment, the compositioncomprising immune effector cells comprising one or more DARIC immunereceptor polypeptides contemplated herein is administered with ananti-inflammatory agent. Anti-inflammatory agents or drugs include, butare not limited to, steroids and glucocorticoids (includingbetamethasone, budesonide, dexamethasone, hydrocortisone acetate,hydrocortisone, hydrocortisone, methylprednisolone, prednisolone,prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs(NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate,sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide andmycophenolate.

Other exemplary NSAIDs are chosen from the group consisting ofibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX®(rofecoxib) and CELEBREX® (celecoxib), and sialylates. Exemplaryanalgesics are chosen from the group consisting of acetaminophen,oxycodone, tramadol of proporxyphene hydrochloride. Exemplaryglucocorticoids are chosen from the group consisting of cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone, orprednisone. Exemplary biological response modifiers include moleculesdirected against cell surface markers (e.g., CD4, CD5, etc.), cytokineinhibitors, such as the TNF antagonists (e.g., etanercept (ENBREL®),adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitorsand adhesion molecule inhibitors. The biological response modifiersinclude monoclonal antibodies as well as recombinant forms of molecules.Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine,methotrexate, penicillamine, leflunomide, sulfasalazine,hydroxychloroquine, Gold (oral (auranofin) and intramuscular) andminocycline.

Illustrative examples of therapeutic antibodies suitable for combinationwith the modified T cells comprising one or more DARIC immune receptorpolypeptides contemplated herein, include but are not limited to,atezolizumab, avelumab, bavituximab, bevacizumab (avastin), bivatuzumab,blinatumomab, conatumumab, daratumumab, duligotumab, dacetuzumab,dalotuzumab, durvalumab, elotuzumab (HuLuc63), gemtuzumab, ibritumomab,indatuximab, inotuzumab, ipilimumab, lorvotuzumab, lucatumumab,milatuzumab, moxetumomab, nivolumab, ocaratuzumab, ofatumumab,pembrolizumab, rituximab, siltuximab, teprotumumab, and ublituximab.

In certain embodiments, the compositions described herein areadministered in conjunction with a cytokine. By “cytokine” as usedherein is meant a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Examplesof such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-beta;platelet-growth factor; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta; insulin-like growth factor-I and —II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-alpha, beta, and -gamma; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12;IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture, and biologically active equivalents of thenative sequence cytokines.

I. Therapeutic Methods

The immune effector cells, including CAR T cells, comprising a DARICimmune receptor contemplated herein provide improved methods of adoptiveimmunotherapy for use in the prevention, treatment, and amelioration of,or for preventing, treating, or ameliorating at least one symptomassociated with, a cancer, GVHD, an infectious disease, an autoimmunedisease, an inflammatory disease, or an immunodeficiency.

The immune effector cells that comprise an engineered receptor and aDARIC immune receptor contemplated herein provide improved drug productsfor use in the prevention, treatment, or amelioration of at least onesymptom of a cancer, GVHD, an infectious disease, an autoimmune disease,an inflammatory disease, or an immunodeficiency. As used herein, theterm “drug product” refers to modified cells produced using thecompositions and methods contemplated herein. In particular embodiments,the drug product comprises genetically modified immune effector cells, Tcells comprising an engineered receptor, or CAR T cells further modifiedto express a DARIC immune receptor. Moreover, the modified T cellscontemplated in particular embodiments provide safer and moreefficacious adoptive cell therapies because they are resistant to T cellexhaustion and display increased durability and persistence in the tumormicroenvironment that can lead to sustained therapy.

In particular embodiments, an effective amount of modified immuneeffector cells or T cells comprising an engineered receptor and a DARICimmune receptor are administered to a subject to prevent, treat, orameliorate at least one symptom of a cancer, GVHD, an infectiousdisease, an autoimmune disease, an inflammatory disease, or animmunodeficiency.

In particular embodiments, a method of preventing, treating, orameliorating at least one symptom of a cancer comprises administeringthe subject an effective amount of modified immune effector cells or Tcells comprising a DARIC immune receptor and an engineered TCR, CAR, orother therapeutic transgene to redirect the cells to a tumor or cancer.The genetically modified cells are a more durable and persistent drugproduct because the cells are more resistant to immunosuppressivesignals from the tumor microenvironment by virtue of transducing achemically regulatable immunostimulatory signal.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of solid tumors orcancers.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of solid tumors or cancersincluding, but not limited to: adrenal cancer, adrenocortical carcinoma,anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoidtumor, basal cell carcinoma, bile duct cancer, bladder cancer, bonecancer, brain/CNS cancer, breast cancer, bronchial tumors, cardiactumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordoma,colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma insitu (DCIS) endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor,extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibroushistiosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumors, gastrointestinal stromal tumor(GIST), germ cell tumors, glioma, glioblastoma, head and neck cancer,hemangioblastoma, hepatocellular cancer, hypopharyngeal cancer,intraocular melanoma, kaposi sarcoma, kidney cancer, laryngeal cancer,leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer,non-small cell lung cancer, lung carcinoid tumor, malignantmesothelioma, medullary carcinoma, medulloblastoma, menangioma,melanoma, Merkel cell carcinoma, midline tract carcinoma, mouth cancer,myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms,nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,neuroblastoma, oligodendroglioma, oral cancer, oral cavity cancer,oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer,pancreatic islet cell tumors, papillary carcinoma, paraganglioma,parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritonealcancer, prostate cancer, rectal cancer, retinoblastoma, renal cellcarcinoma, renal pelvis and ureter cancer, rhabdomyosarcoma, salivarygland cancer, sebaceous gland carcinoma, skin cancer, soft tissuesarcoma, squamous cell carcinoma, small cell lung cancer, smallintestine cancer, stomach cancer, sweat gland carcinoma, synovioma,testicular cancer, throat cancer, thymus cancer, thyroid cancer,urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer,vascular cancer, vulvar cancer, and Wilms Tumor.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of solid tumors or cancersincluding, without limitation, liver cancer, pancreatic cancer, lungcancer, breast cancer, bladder cancer, brain cancer, bone cancer,thyroid cancer, kidney cancer, or skin cancer.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of various cancersincluding but not limited to pancreatic, bladder, and lung.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of liquid cancers orhematological cancers.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of B-cell malignancies,including but not limited to: leukemias, lymphomas, and multiplemyeloma.

In particular embodiments, the modified immune effector cellscontemplated herein are used in the treatment of liquid cancersincluding, but not limited to leukemias, lymphomas, and multiplemyelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia(AML), myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia, hairy cell leukemia (HCL), chronic lymphocytic leukemia(CLL), and chronic myeloid leukemia (CIVIL), chronic myelomonocyticleukemia (CMML) and polycythemia vera, Hodgkin lymphoma, nodularlymphocyte-predominant Hodgkin lymphoma, Burkitt lymphoma, smalllymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicularlymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosisfungoides, anaplastic large cell lymphoma, Sézary syndrome, precursorT-lymphoblastic lymphoma, multiple myeloma, overt multiple myeloma,smoldering multiple myeloma, plasma cell leukemia, non-secretorymyeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma ofbone, and extramedullary plasmacytoma.

Preferred cells for use in the methods contemplated herein includeautologous/autogeneic (“self”) cells, preferably hematopoietic cells,more preferably T cells, and more preferably immune effector cells.

In particular embodiments, methods comprising administering atherapeutically effective amount of modified immune effector cellscontemplated herein or a composition comprising the same, to a patientin need thereof, alone or in combination with one or more therapeuticagents, are provided. In certain embodiments, the cells are used in thetreatment of patients at risk for developing a cancer, GVHD, aninfectious disease, an autoimmune disease, an inflammatory disease, oran immunodeficiency. Thus, particular embodiments comprise the treatmentor prevention or amelioration of at least one symptom of a cancer, aninfectious disease, an autoimmune disease, an inflammatory disease, oran immunodeficiency comprising administering to a subject in needthereof, a therapeutically effective amount of the modified immuneeffector cells contemplated herein.

In one embodiment, a method of treating a cancer, GVHD, an infectiousdisease, an autoimmune disease, an inflammatory disease, or animmunodeficiency in a subject in need thereof comprises administering aneffective amount, e.g., therapeutically effective amount of acomposition comprising modified immune effector cells contemplatedherein. The quantity and frequency of administration will be determinedby such factors as the condition of the patient, and the type andseverity of the patient's disease, although appropriate dosages may bedetermined by clinical trials.

In one illustrative embodiment, the effective amount of modified immuneeffector cells provided to a subject is at least 2×10⁶ cells/kg, atleast 3×10⁶ cells/kg, at least 4×10⁶ cells/kg, at least 5×10⁶ cells/kg,at least 6×10⁶ cells/kg, at least 7×10⁶ cells/kg, at least 8×10⁶cells/kg, at least 9×10⁶ cells/kg, or at least 10×10⁶ cells/kg, or morecells/kg, including all intervening doses of cells.

In another illustrative embodiment, the effective amount of modifiedimmune effector cells provided to a subject is about 2×10⁶ cells/kg,about 3×10⁶ cells/kg, about 4×10⁶ cells/kg, about 5×10⁶ cells/kg, about6×10⁶ cells/kg, about 7×10⁶ cells/kg, about 8×10⁶ cells/kg, about 9×10⁶cells/kg, or about 10×10⁶ cells/kg, or more cells/kg, including allintervening doses of cells.

In another illustrative embodiment, the effective amount of modifiedimmune effector cells provided to a subject is from about 2×10⁶ cells/kgto about 10×10⁶ cells/kg, about 3×10⁶ cells/kg to about 10×10⁶ cells/kg,about 4×10⁶ cells/kg to about 10×10⁶ cells/kg, about 5×10⁶ cells/kg toabout 10×10⁶ cells/kg, 2×10⁶ cells/kg to about 6×10⁶ cells/kg, 2×10⁶cells/kg to about 7×10⁶ cells/kg, 2×10⁶ cells/kg to about 8×10⁶cells/kg, 3×10⁶ cells/kg to about 6×10⁶ cells/kg, 3×10⁶ cells/kg toabout 7×10⁶ cells/kg, 3×10⁶ cells/kg to about 8×10⁶ cells/kg, 4×10⁶cells/kg to about 6×10⁶ cells/kg, 4×10⁶ cells/kg to about 7×10⁶cells/kg, 4×10⁶ cells/kg to about 8×10⁶ cells/kg, 5×10⁶ cells/kg toabout 6×10⁶ cells/kg, 5×10⁶ cells/kg to about 7×10⁶ cells/kg, 5×10⁶cells/kg to about 8×10⁶ cells/kg, or 6×10⁶ cells/kg to about 8×10⁶cells/kg, including all intervening doses of cells.

One of ordinary skill in the art would recognize that multipleadministrations of the compositions contemplated in particularembodiments may be required to effect the desired therapy. For example,a composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10years, or more.

In certain embodiments, it may be desirable to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom, and reinfuse thepatient with these activated and expanded T cells. This process can becarried out multiple times every few weeks. In certain embodiments, Tcells can be activated from blood draws of from 10cc to 400cc. Incertain embodiments, T cells are activated from blood draws of 20cc,30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc,300cc, 350cc, or 400cc or more. Not to be bound by theory, using thismultiple blood draw/multiple reinfusion protocol may serve to select outcertain populations of T cells.

In one embodiment, a method of treating a subject diagnosed with acancer, comprises removing immune effector cells from the subject,modifying the immune effector cells by introducing one or more vectorsencoding an engineered antigen receptor and one or more DARIC immunereceptors and producing a population of modified immune effector cells,and administering the population of modified immune effector cells tothe same subject. In a preferred embodiment, the immune effector cellscomprise T cells.

The methods for administering the cell compositions contemplated inparticular embodiments include any method which is effective to resultin reintroduction of ex vivo modified immune effector cells or onreintroduction of the modified progenitors of immune effector cells thaton introduction into a subject differentiate into mature immune effectorcells. One method comprises modifying peripheral blood T cells ex vivoby introducing one or more vectors encoding an engineered antigenreceptor and one or more DARIC immune receptors and returning thetransduced cells into the subject.

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings contemplated herein that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims. The following examples areprovided by way of illustration only and not by way of limitation. Thoseof skill in the art will readily recognize a variety of noncriticalparameters that could be changed or modified in particular embodimentsto yield essentially similar results.

EXAMPLES Example 1

Rapamycin Induces IFNγ Expression in IL-18 Receptor DARIC T Cells

IL-18 receptor (IL-18R) DARIC binding and signaling components weredesigned, constructed, and verified. An IL-18R DARIC lentiviral vectorwas constructed comprising an MNDU3 promoter operably linked to apolynucleotide encoding: a DARIC signaling component (CD8α-signalpeptide, an FRB variant (T82L), and an IL-18R1 transmembrane andsignaling domain); a P2A sequence; and a DARIC binding component (anIgκ-signal peptide, an FKBP12 domain, and a IL-18RAP transmembrane andsignaling domain). An IL-18R DARIC-GFP lentiviral vector was constructedby appending another polynucleotide encoding a P2A sequence and GFP tothe to the polynucleotide encoding the IL-18R1 signaling domain inaforementioned vector. T cells transduced with the IL-18R DARIC andIL-18R DARIC-GFP lentiviral vectors express the polypeptides shown inFIG. 1A. See, e.g., SEQ ID NOs: 1-4.

GFP expression was measured in donor PBMCs transduced with LVV encodingIL-18R-DARIC-GFP to assess transduction efficiency. FIG. 1B.

Cell growth was measured in donor PBMCs transduced with LVV encodingIL-18R-DARIC-GFP. The transduced cells showed similar growth kinetics toUntransduced control PBMCs. FIG. 1C.

CD62L and CD45Ra expression were measure in donor PBMCs transduced withLVV encoding IL-18R-DARIC-GFP. T cell subset development as assessed byCD62L and CD45Ra expression was similar among IL-18R-DARIC-GFPtransduced cells and untransduced controls. FIG. 1D.

Donor PBMCs were transduced with LVV encoding IL-18R-DARIC-GFP.Transduced PBMCs and untransduced control PBMCs were pre-treated with 50ng/mL human recombinant IL-12 for 24 hours. The treated cells werewashed and cultured in medium only, medium with 100 ng/mL humanrecombinant IL-18 or medium with 1 nM rapamycin. T cells cultured inIL-18 showed increased IFNγ production in both untransduced controls andcells transduced with IL-18R-DARIC-GFP. In contrast, only cellstransduced with IL-18R-DARIC-GFP showed increased IFNγ production whentreated with rapamycin. FIG. 2.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific embodiments disclosed inthe specification and the claims, but should be construed to include allpossible embodiments along with the full scope of equivalents to whichsuch claims are entitled. Accordingly, the claims are not limited by thedisclosure.

1. A fusion polypeptide comprising: (a) a first polypeptide comprising:(i) a first multimerization domain; (ii) a first transmembrane domain;and (iii) a first immune receptor intracellular signaling domain; (b) apolypeptide cleavage signal; and (c) a second polypeptide comprising:(i) a second multimerization domain; (ii) a second transmembrane domain;and (iii) a second immune receptor intracellular signaling domain. 2.The fusion polypeptide of claim 1, wherein the first multimerizationdomain and the second multimerization domain are the same.
 3. The fusionpolypeptide of claim 1, wherein the first multimerization domain and thesecond multimerization domain are different.
 4. The fusion polypeptideof any one of claims 1 to 3, wherein the first multimerization domainand the second multimerization domain associate with a bridging factorselected from the group consisting of: rapamycin or a rapalog thereof,coumermycin or a derivative thereof, gibberellin or a derivativethereof, abscisic acid (ABA) or a derivative thereof, methotrexate or aderivative thereof, cyclosporin A or a derivative thereof,FK506/cyclosporin A (FKCsA) or a derivative thereof, and trimethoprim(Tmp)-synthetic ligand for FK506 binding protein (FKBP) (SLF) or aderivative thereof.
 5. The fusion polypeptide of any one of claims 1 to4, wherein the first multimerization domain and the secondmultimerization domain are a pair selected from the group consisting of:FKBP and FKBP12-rapamycin binding (FRB) or variants thereof; FKBP andcalcineurin or variants thereof; FKBP and cyclophilin or variantsthereof; FKBP and bacterial dihydrofolate reductase (DHFR) or variantsthereof; calcineurin and cyclophilin or variants thereof; PYR1-like 1(PYL1) and abscisic acid insensitive 1 (ABI1) or variants thereof; andGIB1 and GAI or variants thereof.
 6. The fusion polypeptide of any oneof claims 1 to 5, wherein the first multimerization domain comprises aFKBP polypeptide or variant thereof, and the second multimerizationdomain comprises a FRB polypeptide or variant thereof.
 7. The fusionpolypeptide of any one of claims 1 to 5, wherein the firstmultimerization domain comprises a FRB polypeptide or variant thereof,and the second multimerization domain comprises a FKBP polypeptide orvariant thereof.
 8. The fusion polypeptide of any one of claims 1 to 5,wherein the first and second multimerization domains are selected fromFRB T2098L and FKBP12; and the bridging factor is AP21967.
 9. The fusionpolypeptide of any one of claims 4 to 7, wherein the bridging factor isselected from the group consisting of: AP21967, sirolimus, everolimus,novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus,umirolimus, and zotarolimus.
 10. The fusion polypeptide of any one ofclaims 1 to 9, wherein the first transmembrane domain and the secondtransmembrane domain are independently selected from the groupconsisting of: a CD4 transmembrane domain, a CD8α transmembrane domain,an amnionless (AMN) transmembrane domain, a CD28 transmembrane domain, aCD154 transmembrane domain, and a CD71 transmembrane domain.
 11. Thefusion polypeptide of any one of claims 1 to 10, wherein the firsttransmembrane domain and the second transmembrane domain areindependently selected from the group consisting of: a CD4 transmembranedomain and a CD8α transmembrane domain.
 12. The fusion polypeptide ofany one of claims 1 to 11, wherein the first transmembrane domain andthe second transmembrane domain are the same.
 13. The fusion polypeptideof any one of claims 1 to 11, wherein the first transmembrane domain andthe second transmembrane domain are different.
 14. The fusionpolypeptide of any one of claims 1 to 13, wherein first immune receptorintracellular signaling domain and the second immune receptorintracellular signaling domain are isolated from a cytokine receptor, aninterleukin receptor, a pattern recognition receptor, or a toll-likereceptor.
 15. The fusion polypeptide of any one of claims 1 to 14,wherein the first immune receptor intracellular signaling domaincomprises an IL-12Rβ2 intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an IL-12Rβ1intracellular signaling domain.
 16. The fusion polypeptide of any one ofclaims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an IL-12Rβ1 intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-12Rβ2 intracellular signaling domain.
 17. The fusion polypeptideof any one of claims 1 to 14, wherein the first immune receptorintracellular signaling domain comprises an IL-7Rα intracellularsignaling domain and the second immune receptor intracellular signalingdomain comprises an IL-2Rγ intracellular signaling domain.
 18. Thefusion polypeptide of any one of claims 1 to 14, wherein the firstimmune receptor intracellular signaling domain comprises an IL-2Rγintracellular signaling domain and the second immune receptorintracellular signaling domain comprises an IL-7Rα intracellularsignaling domain.
 19. The fusion polypeptide of any one of claims 1 to14, wherein the first immune receptor intracellular signaling domaincomprises an IL-2Rβ intracellular signaling domain and the second immunereceptor intracellular signaling domain comprises an IL-2Rγintracellular signaling domain.
 20. The fusion polypeptide of any one ofclaims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rβ intracellular signaling domain.
 21. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an IL-21R intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rγ intracellular signaling domain.
 22. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-21R intracellular signaling domain.
 23. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an IL-18R1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-18RAP intracellular signaling domain.
 24. The fusion polypeptide ofany one of claims 1 to 14, wherein the first immune receptorintracellular signaling domain comprises an IL-18RAP intracellularsignaling domain and the second immune receptor intracellular signalingdomain comprises an IL-18R1 intracellular signaling domain.
 25. Thefusion polypeptide of any one of claims 1 to 14, wherein the firstimmune receptor intracellular signaling domain comprises an IL-1R1intracellular signaling domain and the second immune receptorintracellular signaling domain comprises an IL-1RAP intracellularsignaling domain.
 26. The fusion polypeptide of any one of claims 1 to14, wherein the first immune receptor intracellular signaling domaincomprises an IL-1RAP intracellular signaling domain and the secondimmune receptor intracellular signaling domain comprises an IL-1R1intracellular signaling domain.
 27. The fusion polypeptide of any one ofclaims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an IL-1RL2 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1RAP intracellular signaling domain.
 28. The fusion polypeptide ofany one of claims 1 to 14, wherein the first immune receptorintracellular signaling domain comprises an IL-1RAP intracellularsignaling domain and the second immune receptor intracellular signalingdomain comprises an IL-1RL2 intracellular signaling domain.
 29. Thefusion polypeptide of any one of claims 1 to 14, wherein the firstimmune receptor intracellular signaling domain comprises an IFNAR1intracellular signaling domain and the second immune receptorintracellular signaling domain comprises an IFNAR2 intracellularsignaling domain.
 30. The fusion polypeptide of any one of claims 1 to14, wherein the first immune receptor intracellular signaling domaincomprises an IFNAR2 intracellular signaling domain and the second immunereceptor intracellular signaling domain comprises an IFNAR1intracellular signaling domain.
 31. The fusion polypeptide of any one ofclaims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR1 intracellular signaling domain.
 32. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR2 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR2 intracellular signaling domain.
 33. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR3 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR3 intracellular signaling domain.
 34. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR4 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR4 intracellular signaling domain.
 35. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR5 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR5 intracellular signaling domain.
 36. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR6 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR6 intracellular signaling domain.
 37. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR7 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR7 intracellular signaling domain.
 38. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR8 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR8 intracellular signaling domain.
 39. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR9 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR9 intracellular signaling domain.
 40. The fusion polypeptide of anyone of claims 1 to 14, wherein the first immune receptor intracellularsignaling domain comprises an TLR10 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR10 intracellular signaling domain.
 41. The fusion polypeptide of anyone of claims 1 to 40, wherein the polypeptide cleavage signal is aviral self-cleaving polypeptide.
 42. The fusion polypeptide of any oneof claims 1 to 41, wherein the polypeptide cleavage signal is a viralself-cleaving 2A polypeptide.
 43. The fusion polypeptide of any one ofclaims 1 to 42, wherein the polypeptide cleavage signal is a viralself-cleaving polypeptide selected from the group consisting of: afoot-and-mouth disease virus (FMDV) (F2A) peptide, an equine rhinitis Avirus (ERAV) (E2A) peptide, a Thosea asigna virus (TaV) (T2A) peptide, aporcine teschovirus-1 (PTV-1) (P2A) peptide, a Theilovirus 2A peptide,and an encephalomyocarditis virus 2A peptide.
 44. The fusion polypeptideof any one of claims 1 to 43, wherein the first multimerization domainlocalizes extracellularly when the first polypeptide is expressed andthe second multimerization domain localizes extracellularly when thesecond polypeptide is expressed.
 45. A composition comprising the fusionpolypeptide of any one of claims 1 to
 44. 46. A composition comprising:(a) a first polypeptide comprising: (i) a first multimerization domain;(ii) a first transmembrane domain; and (iii) a first immune receptorintracellular signaling domain; and (b) a second polypeptide comprising:(i) a second multimerization domain; (ii) a second transmembrane domain;and (iii) a second immune receptor intracellular signaling domain. 47.The composition of claim 46, wherein the first multimerization domainand the second multimerization domain are the same.
 48. The compositionof claim 46, wherein the first multimerization domain and the secondmultimerization domain are different.
 49. The composition of any one ofclaims 46 to 48, wherein the first multimerization domain and the secondmultimerization domain associate with a bridging factor selected fromthe group consisting of: rapamycin or a rapalog thereof, coumermycin ora derivative thereof, gibberellin or a derivative thereof, abscisic acid(ABA) or a derivative thereof, methotrexate or a derivative thereof,cyclosporin A or a derivative thereof, FK506/cyclosporin A (FKCsA) or aderivative thereof, and trimethoprim (Tmp)-synthetic ligand for FK506binding protein (FKBP) (SLF) or a derivative thereof.
 50. Thecomposition of any one of claims 46 to 49, wherein the firstmultimerization domain and the second multimerization domain are a pairselected from the group consisting of: FKBP and FKBP12-rapamycin binding(FRB) or variants thereof; FKBP and calcineurin or variants thereof;FKBP and cyclophilin or variants thereof; FKBP and bacterialdihydrofolate reductase (DHFR) or variants thereof; calcineurin andcyclophilin or variants thereof; PYR1-like 1 (PYL1) and abscisic acidinsensitive 1 (ABI1) or variants thereof; and GIB1 and GAI or variantsthereof.
 51. The composition of any one of claims 46 to 50, wherein thefirst multimerization domain comprises a FKBP polypeptide or variantthereof, and the second multimerization domain comprises a FRBpolypeptide or variant thereof.
 52. The composition of any one of claims46 to 50, wherein the first multimerization domain comprises a FRBpolypeptide or variant thereof, and the second multimerization domaincomprises a FKBP polypeptide or variant thereof.
 53. The composition ofany one of claims 46 to 50, wherein the first and second multimerizationdomains are selected from FRB T2098L and FKBP12; and the bridging factoris AP21967.
 54. The composition of any one of claims 49 to 52, whereinthe bridging factor is selected from the group consisting of: AP21967,sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus,tacrolimus, temsirolimus, umirolimus, and zotarolimus.
 55. Thecomposition of any one of claims 46 to 54, wherein the firsttransmembrane domain and the second transmembrane domain areindependently selected from the group consisting of: a CD4 transmembranedomain, a CD8α transmembrane domain, an amnionless (AMN) transmembranedomain, a CD28 transmembrane domain, a CD154 transmembrane domain, and aCD71 transmembrane domain.
 56. The composition of any one of claims 46to 55, wherein the first transmembrane domain and the secondtransmembrane domain are independently selected from the groupconsisting of: a CD4 transmembrane domain and a CD8α transmembranedomain.
 57. The composition of any one of claims 46 to 56, wherein thefirst transmembrane domain and the second transmembrane domain are thesame.
 58. The composition of any one of claims 46 to 56, wherein thefirst transmembrane domain and the second transmembrane domain aredifferent.
 59. The composition of any one of claims 46 to 58, whereinfirst immune receptor intracellular signaling domain and the secondimmune receptor intracellular signaling domain are isolated from acytokine receptor, an interleukin receptor, a pattern recognitionreceptor, or a toll-like receptor.
 60. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-12Rβ2 intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-12Rβ1 intracellular signaling domain.
 61. The composition of anyone of claims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-12Rβ1 intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-12Rβ2 intracellular signaling domain.
 62. The composition of anyone of claims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-7Rα intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rγ intracellular signaling domain.
 63. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-7Rα intracellular signaling domain.
 64. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-2Rβ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rγ intracellular signaling domain.
 65. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rβ intracellular signaling domain.
 66. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-21R intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-2Rγ intracellular signaling domain.
 67. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-2Rγ intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-21R intracellular signaling domain.
 68. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-18R1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-18RAP intracellular signaling domain.
 69. The composition of any oneof claims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-18RAP intracellular signaling domainand the second immune receptor intracellular signaling domain comprisesan IL-18R1 intracellular signaling domain.
 70. The composition of anyone of claims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-1R1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1RAP intracellular signaling domain.
 71. The composition of any oneof claims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-1RAP intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1R1 intracellular signaling domain.
 72. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-1RL2 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1RAP intracellular signaling domain.
 73. The composition of any oneof claims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IL-1RAP intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIL-1RL2 intracellular signaling domain.
 74. The composition of any oneof claims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IFNAR1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIFNAR2 intracellular signaling domain.
 75. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an IFNAR2 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anIFNAR1 intracellular signaling domain.
 76. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR1 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR1 intracellular signaling domain.
 77. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR2 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR2 intracellular signaling domain.
 78. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR3 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR3 intracellular signaling domain.
 79. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR4 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR4 intracellular signaling domain.
 80. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR5 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR5 intracellular signaling domain.
 81. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR6 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR6 intracellular signaling domain.
 82. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR7 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR7 intracellular signaling domain.
 83. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR8 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR8 intracellular signaling domain.
 84. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR9 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR9 intracellular signaling domain.
 85. The composition of any one ofclaims 46 to 59, wherein the first immune receptor intracellularsignaling domain comprises an TLR10 intracellular signaling domain andthe second immune receptor intracellular signaling domain comprises anTLR10 intracellular signaling domain.
 86. The compositions of any one ofclaims 45 to 85, further comprising a cell.
 87. A polypeptide complexcomprising the first and second polypeptides of any one of claims 1 to85.
 88. A polynucleotide encoding the fusion polypeptide of any one ofclaims 1 to 45 or the first and second polypeptides of any one of claims1 to
 85. 89. A cDNA encoding the fusion polypeptide of any one of claims1 to 45 or the first and second polypeptides of any one of claims 1 to85.
 90. An RNA encoding the fusion polypeptide of any one of claims 1 to45 or the first and second polypeptides of any one of claims 1 to 85.91. A vector comprising the polynucleotide of any one of claims 88 to90.
 92. A cell comprising the fusion polypeptide of any one of claims 1to 45, the first and second polypeptides of any one of claims 1 to 85,the polynucleotide of any one of claims 88-90, or the vector of claim91.
 93. The cell of claim 92, wherein the cell is a hematopoietic cell.94. The cell of claim 92 or 93, wherein the cell is a T cell.
 95. Thecell of any one of claims 92 to 94, wherein the cell is a CD3⁺, CD4⁺,and/or CD8⁺ cell.
 96. The cell of any one of claims 92 to 95, whereinthe cell is an immune effector cell.
 97. The cell of any one of claims92 to 96, wherein the cell is a cytotoxic T lymphocytes (CTLs), a tumorinfiltrating lymphocytes (TILs), or a helper T cells.
 98. The cell ofany one of claims 92 to 96, wherein the cell is a natural killer (NK)cell or natural killer T (NKT) cell.
 99. The cell of any one of claims92 to 98, wherein the source of the cell is peripheral blood mononuclearcells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissuefrom a site of infection, ascites, pleural effusion, spleen tissue, ortumors.
 100. The cell of any one of claims 92 to 99, further comprisingan engineered antigen receptor.
 101. The cell of claim 100, wherein theengineered antigen receptor is selected from the group consisting of: anengineered T cell receptor (TCR), a chimeric antigen receptor (CAR), aDARIC receptor or components thereof, and a chimeric cytokine receptor.102. A composition comprising the fusion polypeptide of any one ofclaims 1 to 45, the first and second polypeptides of any one of claims 1to 85, the polynucleotide of any one of claims 88 to 90, the vector ofclaim 91, or the cell of any one of claims 92 to
 101. 103. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the fusion polypeptide of any one of claims 1 to 45, thefirst and second polypeptides of any one of claims 1 to 85, thepolynucleotide of any one of claims 88 to 90, the vector of claim 91, orthe cell of any one of claims 92 to
 101. 104. A method of treating asubject in need thereof comprising administering the subject aneffective amount of the composition of claim
 103. 105. A method oftreating, preventing, or ameliorating at least one symptom of a cancer,infectious disease, autoimmune disease, inflammatory disease, andimmunodeficiency, or condition associated therewith, comprisingadministering to the subject an effective amount of the composition ofclaim
 103. 106. A method of treating a solid cancer comprisingadministering to the subject an effective amount of the composition ofclaim
 103. 107. The method of claim 106, wherein the solid cancercomprises liver cancer, pancreatic cancer, lung cancer, breast cancer,ovarian cancer, prostate cancer, testicular cancer, bladder cancer,brain cancer, sarcoma, head and neck cancer, bone cancer, thyroidcancer, kidney cancer, or skin cancer.
 108. The method of claim 105 or106, wherein the solid cancer is a pancreatic cancer, a lung cancer, ora breast cancer.
 109. A method of treating a hematological malignancycomprising administering to the subject an effective amount of thecomposition of claim
 103. 110. The method of claim 109, wherein thehematological malignancy is a leukemia, lymphoma, or multiple myeloma.